PMC 20201216 pmc.key 4746701 CC BY no 0 0 10.1038/srep20261 srep20261 4746701 27064360 20261 This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ surname:Jeong;given-names:Hanbin surname:Sim;given-names:Hyo Jung surname:Song;given-names:Eun Kyung surname:Lee;given-names:Hakbong surname:Ha;given-names:Sung Chul surname:Jun;given-names:Youngsoo surname:Park;given-names:Tae Joo surname:Lee;given-names:Changwook TITLE front 6 2016 0 Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway 0.9988518 evidence cleaner0 2023-07-26T11:52:54Z DUMMY: Crystal structure 0.9992508 protein cleaner0 2023-07-26T08:23:58Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:31:35Z SLR ABSTRACT abstract 81 Terminally misfolded proteins are selectively recognized and cleared by the endoplasmic reticulum-associated degradation (ERAD) pathway. SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery. 0.9992866 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.99754673 evidence cleaner0 2023-07-26T11:52:55Z DUMMY: crystal structure 0.9909797 taxonomy_domain cleaner0 2023-07-26T08:29:39Z DUMMY: mouse 0.9992374 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.9963008 structure_element cleaner0 2023-07-26T09:47:28Z SO: central domain 0.99568605 structure_element cleaner0 2023-07-26T08:25:29Z SO: Sel1-Like Repeats 0.9770133 structure_element cleaner0 2023-07-26T08:25:35Z SO: SLR motifs 5 to 9 0.51770043 structure_element cleaner0 2023-07-26T08:23:44Z SO: SEL1Lcent 0.98432606 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.99863064 oligomeric_state cleaner0 2023-07-26T08:24:28Z DUMMY: homodimer 0.9969376 protein_state cleaner0 2023-07-26T08:24:19Z DUMMY: head-to-tail 0.996372 structure_element cleaner0 2023-07-26T08:25:41Z SO: SLR motif 9 0.9987109 oligomeric_state cleaner0 2023-07-26T08:24:33Z DUMMY: dimer 0.917501 protein_state cleaner0 2023-07-26T09:03:04Z DUMMY: domain-swapped 0.9982927 site cleaner0 2023-07-26T12:02:50Z SO: dimeric interface 0.9991167 protein_state cleaner0 2023-07-26T08:24:13Z DUMMY: full-length 0.9992772 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L oligomeric_state DUMMY: cleaner0 2023-07-26T08:25:00Z self-oligomer 0.526936 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.9985261 taxonomy_domain cleaner0 2023-07-26T08:25:51Z DUMMY: mammalian 0.99810743 structure_element cleaner0 2023-07-26T08:33:21Z SO: SLR-C 0.9958229 structure_element cleaner0 2023-07-26T08:33:26Z SO: SLR motifs 10 and 11 0.9992606 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T09:51:11Z luminal loops 0.9960486 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 structure_element SO: cleaner0 2023-07-26T08:31:35Z SLR 0.9993025 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L INTRO paragraph 1119 Protein quality control in the endoplasmic reticulum (ER) is essential for maintenance of cellular homeostasis in eukaryotes and is implicated in many severe diseases. Terminally misfolded proteins in the lumen or membrane of the ER are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. The process is called ER-associated protein degradation (ERAD) and is conserved in all eukaryotes. Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. The components are differentially localized from the lumen and membrane of the ER to the cytosol, and have different functions in the ERAD process. Yeast ERAD components, which have been extensively characterized through genetic and biochemical studies, are comparable with mammalian ERAD components, sharing similar molecular functions and structural composition. 0.9985447 taxonomy_domain cleaner0 2023-07-26T08:25:56Z DUMMY: eukaryotes 0.9758303 protein_state cleaner0 2023-07-26T11:59:39Z DUMMY: polyubiquitinated 0.9492112 complex_assembly cleaner0 2023-07-26T12:05:11Z GO: proteasome 0.9905149 protein_state cleaner0 2023-07-26T11:59:44Z DUMMY: conserved 0.9946179 taxonomy_domain cleaner0 2023-07-26T08:25:57Z DUMMY: eukaryotes 0.9990409 protein cleaner0 2023-07-26T08:26:24Z PR: HRD1 0.99905616 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.99904174 protein cleaner0 2023-07-26T08:26:29Z PR: Hrd3p 0.9990537 protein cleaner0 2023-07-26T08:26:37Z PR: Derlin-1, -2, -3 0.99914646 protein cleaner0 2023-07-26T08:26:42Z PR: Der1p 0.99912745 protein cleaner0 2023-07-26T08:26:53Z PR: HERP-1, -2 0.9991105 protein cleaner0 2023-07-26T08:26:59Z PR: Usa1p 0.99894625 protein cleaner0 2023-07-26T08:27:04Z PR: OS9 0.99881977 protein cleaner0 2023-07-26T08:27:09Z PR: Yos9 0.99912435 protein cleaner0 2023-07-26T08:27:13Z PR: XTP-B 0.9990497 protein cleaner0 2023-07-26T08:27:19Z PR: Grp94 0.99891496 taxonomy_domain cleaner0 2023-07-26T08:27:25Z DUMMY: yeast 0.99853647 taxonomy_domain cleaner0 2023-07-26T08:27:32Z DUMMY: metazoans 0.99890244 taxonomy_domain cleaner0 2023-07-26T08:27:26Z DUMMY: Yeast 0.9988557 experimental_method cleaner0 2023-07-26T12:13:45Z MESH: genetic and biochemical studies 0.9987692 taxonomy_domain cleaner0 2023-07-26T08:25:52Z DUMMY: mammalian INTRO paragraph 2186 The HRD1 E3 ubiquitin ligase, which is embedded in the ER membrane, is involved in translocating ERAD substrates across the ER membrane and catalyzing substrate ubiquitination via its cytosolic RING finger domain. SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded. In particular, SEL1L is crucial for translocation of Class I major histocompatibility complex (MHC) heavy chains (HCs). Recent research based on the inducible Sel1l knockout mouse model highlights the physiological functions of SEL1L. SEL1L is required for ER homeostasis, which is essential for protein translation, pancreatic function, and cellular and organismal survival. However, despite the functional importance of SEL1L, the molecular structure of SEL1L has not been solved. Previous biochemical studies reveal that SEL1L is a type I transmembrane protein and has a large luminal domain comprising sets of repeated Sel1-like (SLR) motifs. The SLR motif is a structural motif that closely resembles the tetratricopeptide-repeat (TPR) motif, which is a protein-protein interaction module. Although there is evidence that the luminal domain of SEL1L is involved in substrate recognition or in forming complexes with chaperones, it is not known how the unique structure of the repeated SLR motifs contributes to the molecular function of the HRD1-SEL1L E3 ligase complex and affects ERAD at the molecular level. Furthermore, while repeated SLR motifs are commonly found in tandem arrays, the SLR motifs in SEL1L are, according to the primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters. Therefore, the way in which these unique structural features of SEL1L are related to its critical function in ERAD remains to be elucidated. 0.99861705 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.9934304 protein_type cleaner0 2023-07-26T08:27:55Z MESH: E3 ubiquitin ligase 0.9971369 structure_element cleaner0 2023-07-26T08:28:08Z SO: RING finger domain 0.9992901 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.998187 taxonomy_domain cleaner0 2023-07-26T08:25:52Z DUMMY: mammalian 0.99932337 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p 0.998701 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.9979652 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.9981773 protein_type cleaner0 2023-07-26T12:25:11Z MESH: lectin 0.9990397 protein cleaner0 2023-07-26T08:27:05Z PR: OS9 0.9991423 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L complex_assembly GO: cleaner0 2023-07-26T08:28:59Z Class I major histocompatibility complex 0.9961902 complex_assembly cleaner0 2023-07-26T12:05:21Z GO: MHC 0.703586 protein_type cleaner0 2023-07-26T08:29:20Z MESH: heavy chains 0.958494 protein_type cleaner0 2023-07-26T08:29:31Z MESH: HCs 0.5361406 gene cleaner0 2023-07-26T08:30:21Z GENE: Sel1l experimental_method MESH: cleaner0 2023-07-26T08:30:54Z knockout mouse 0.99895835 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.99898237 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.99911755 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.5043668 evidence cleaner0 2023-07-26T11:53:04Z DUMMY: structure 0.9992186 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.9939858 experimental_method cleaner0 2023-07-26T12:13:52Z MESH: biochemical studies 0.99927205 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.9878181 protein_type cleaner0 2023-07-26T08:31:16Z MESH: type I transmembrane protein 0.992851 structure_element cleaner0 2023-07-26T08:31:23Z SO: luminal domain 0.95882595 structure_element cleaner0 2023-07-26T08:31:31Z SO: repeated Sel1-like 0.98645127 structure_element cleaner0 2023-07-26T08:31:35Z SO: SLR structure_element SO: cleaner0 2023-07-26T08:31:35Z SLR 0.9975922 structure_element cleaner0 2023-07-26T09:44:59Z SO: tetratricopeptide-repeat 0.99426264 structure_element cleaner0 2023-07-26T09:43:43Z SO: TPR 0.99419355 structure_element cleaner0 2023-07-26T08:31:24Z SO: luminal domain 0.9992374 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.82589394 protein_type cleaner0 2023-07-26T11:53:10Z MESH: chaperones structure_element SO: cleaner0 2023-07-26T08:31:35Z SLR 0.9983583 complex_assembly cleaner0 2023-07-26T08:32:42Z GO: HRD1-SEL1L 0.78861254 protein_type cleaner0 2023-07-26T08:32:48Z MESH: E3 ligase structure_element SO: cleaner0 2023-07-26T08:31:35Z SLR structure_element SO: cleaner0 2023-07-26T08:31:35Z SLR 0.99923146 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.9991757 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.99028516 structure_element cleaner0 2023-07-26T08:31:24Z SO: luminal domain structure_element SO: cleaner0 2023-07-26T08:31:35Z SLR 0.9992945 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L INTRO paragraph 4073 To clearly understand the biochemical role of the SLR domains of SEL1L in ERAD, we determined the crystal structure of the central SLR domain of SEL1L. We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1Lcent), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9. We also found that SLR-C, consisting of SLR motifs 10 and 11, directly interacts with the N-terminus luminal loop of HRD1. Based on these observations, we propose a model wherein the SLR domains of SEL1L contribute to the formation of stable oligomers of the ERAD translocation machinery, which is indispensable for ERAD. 0.9992932 structure_element cleaner0 2023-07-26T08:31:35Z SO: SLR 0.99931407 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.9978889 evidence cleaner0 2023-07-26T11:52:55Z DUMMY: crystal structure 0.99859124 structure_element cleaner0 2023-07-26T08:31:35Z SO: SLR 0.9993007 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.9929137 structure_element cleaner0 2023-07-26T09:47:38Z SO: central domain 0.99927694 protein cleaner0 2023-07-26T08:23:59Z PR: SEL1L 0.8446561 structure_element cleaner0 2023-07-26T09:47:43Z SO: SLR motifs 5 through 9 0.9971794 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.9976841 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9741096 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.8348906 structure_element cleaner0 2023-07-26T08:33:22Z SO: SLR-C 0.9868504 structure_element cleaner0 2023-07-26T08:33:26Z SO: SLR motifs 10 and 11 0.99824077 structure_element cleaner0 2023-07-26T09:47:48Z SO: luminal loop 0.9978137 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.9992292 structure_element cleaner0 2023-07-26T08:31:36Z SO: SLR 0.9993494 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.9854754 protein_state cleaner0 2023-07-26T11:59:55Z DUMMY: stable 0.99838114 oligomeric_state cleaner0 2023-07-26T11:55:49Z DUMMY: oligomers RESULTS title_1 4723 Results RESULTS title_2 4731 Structure Determination of SEL1Lcent 0.99846506 experimental_method cleaner0 2023-07-26T12:13:58Z MESH: Structure Determination 0.998895 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent RESULTS paragraph 4768 The Mus musculus SEL1L protein contains 790 amino acids and has 17% sequence identity to its yeast homolog, Hrd3p. Mouse SEL1L contains a fibronectin type II domain at the N-terminus, followed by 11 SLR motifs and a single transmembrane domain at the C-terminus (Fig. 1A). The 11 SLR motifs are located in the ER lumen and account for more than two thirds of the mass of full-length SEL1L. The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735–755), by expression in bacteria. However, the full-length SEL1L protein aggregated in solution and produced no soluble protein. To identify a soluble form of SEL1L, we generated serial truncation constructs of SEL1L based on the predicted SLR motifs and highly conserved regions across several different species. Both SLR-N (residues 194–343) and SLR-C (residues 639–719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography. However, the central region of SEL1L, comprising residues 337–554, was soluble and homogenous in size, as determined by size-exclusion chromatography. To define compact domain boundaries for the central region of SEL1L, we digested the protein with trypsin and analyzed the proteolysis products by SDS-PAGE and N-terminal sequencing. The results of this preliminary biochemical analysis suggested that SEL1L residues 348–533 (SEL1Lcent) would be amenable to structural analysis (Fig. 1A). 0.99817586 species cleaner0 2023-07-26T08:34:14Z MESH: Mus musculus 0.99881727 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.9986405 taxonomy_domain cleaner0 2023-07-26T08:27:26Z DUMMY: yeast 0.9986916 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p 0.99675995 taxonomy_domain cleaner0 2023-07-26T08:29:39Z DUMMY: Mouse 0.9990717 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.9988529 structure_element cleaner0 2023-07-26T09:47:57Z SO: fibronectin type II domain structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR structure_element SO: cleaner0 2023-07-26T08:38:12Z transmembrane domain structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9991053 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.99896204 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9866763 structure_element cleaner0 2023-07-26T08:35:21Z SO: linker sequences structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9888961 structure_element cleaner0 2023-07-26T08:35:33Z SO: SLR-N 0.9852956 structure_element cleaner0 2023-07-26T08:35:37Z SO: SLR motifs 1 to 4 0.99116176 structure_element cleaner0 2023-07-26T08:35:41Z SO: SLR-M 0.93657416 structure_element cleaner0 2023-07-26T08:25:36Z SO: SLR motifs 5 to 9 0.9188196 structure_element cleaner0 2023-07-26T08:33:22Z SO: SLR-C 0.98369825 structure_element cleaner0 2023-07-26T09:48:02Z SO: SLR motifs 10 to 11 0.99843454 experimental_method cleaner0 2023-07-26T12:14:02Z MESH: Sequence alignment structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.8135656 structure_element cleaner0 2023-07-26T08:35:28Z SO: linker sequence 0.9977494 residue_range cleaner0 2023-07-26T08:36:20Z DUMMY: 336–345 0.99132603 structure_element cleaner0 2023-07-26T08:35:33Z SO: SLR-N 0.9862742 structure_element cleaner0 2023-07-26T08:35:41Z SO: SLR-M 0.95723855 structure_element cleaner0 2023-07-26T08:35:28Z SO: linker sequence 0.9976837 residue_range cleaner0 2023-07-26T08:36:26Z DUMMY: 528–635 0.98249346 structure_element cleaner0 2023-07-26T08:35:41Z SO: SLR-M 0.9578497 structure_element cleaner0 2023-07-26T08:33:22Z SO: SLR-C 0.99900556 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.9963148 taxonomy_domain cleaner0 2023-07-26T08:29:39Z DUMMY: mouse 0.9991549 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.99889475 structure_element cleaner0 2023-07-26T08:38:11Z SO: transmembrane domain 0.9977551 residue_range cleaner0 2023-07-26T08:36:22Z DUMMY: 735–755 0.9981437 experimental_method cleaner0 2023-07-26T12:14:12Z MESH: expression in bacteria 0.9991067 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.99904925 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.9991609 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.9660775 experimental_method cleaner0 2023-07-26T12:14:18Z MESH: serial truncation constructs 0.99915016 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9989929 protein_state cleaner0 2023-07-26T08:36:14Z DUMMY: highly conserved 0.88397986 structure_element cleaner0 2023-07-26T08:35:33Z SO: SLR-N 0.997901 residue_range cleaner0 2023-07-26T08:36:28Z DUMMY: 194–343 0.83972365 structure_element cleaner0 2023-07-26T08:33:22Z SO: SLR-C 0.9978097 residue_range cleaner0 2023-07-26T08:36:31Z DUMMY: 639–719 experimental_method MESH: cleaner0 2023-07-26T08:38:02Z MBP tag at the N-terminus 0.998687 experimental_method cleaner0 2023-07-26T12:14:24Z MESH: size-exclusion chromatography structure_element SO: cleaner0 2023-07-26T08:36:53Z central region 0.9990677 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.9978606 residue_range cleaner0 2023-07-26T08:36:34Z DUMMY: 337–554 0.9987895 experimental_method cleaner0 2023-07-26T12:14:29Z MESH: size-exclusion chromatography structure_element SO: cleaner0 2023-07-26T08:36:54Z central region 0.9991781 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L experimental_method MESH: cleaner0 2023-07-26T08:37:23Z digested the protein with trypsin 0.9988027 experimental_method cleaner0 2023-07-26T12:14:37Z MESH: SDS-PAGE 0.99544513 experimental_method cleaner0 2023-07-26T12:14:41Z MESH: N-terminal sequencing 0.99861073 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.99793893 residue_range cleaner0 2023-07-26T08:38:17Z DUMMY: 348–533 0.91120136 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent experimental_method MESH: cleaner0 2023-07-26T08:37:44Z structural analysis RESULTS paragraph 6731 Crystals of SEL1Lcent grew in space group P21 with four copies of SEL1Lcent (a total of 82 kDa) in the asymmetric unit. The structure was determined by the single-wavelength anomalous diffraction (SAD) method using selenium as the anomalous scatterer (Table 1 and Methods). The assignment of residues during model building was aided by the selenium atom positions, and the structure was refined with native data to 2.6 Å resolution with Rwork/Rfree values of 20.7/27.7%. Statistics for data collection and refinement are presented in Table 1. 0.99207085 evidence cleaner0 2023-07-26T11:53:16Z DUMMY: Crystals 0.9989442 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.99910873 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.9972408 evidence cleaner0 2023-07-26T11:53:20Z DUMMY: structure 0.99886435 experimental_method cleaner0 2023-07-26T12:14:47Z MESH: single-wavelength anomalous diffraction 0.99847096 experimental_method cleaner0 2023-07-26T12:14:50Z MESH: SAD 0.9987256 chemical cleaner0 2023-07-26T08:38:44Z CHEBI: selenium 0.99861145 chemical cleaner0 2023-07-26T08:38:44Z CHEBI: selenium 0.9973163 evidence cleaner0 2023-07-26T11:53:28Z DUMMY: structure 0.76061416 evidence cleaner0 2023-07-26T11:53:31Z DUMMY: Rwork/Rfree RESULTS title_2 7279 Overall Structure of SEL1Lcent 0.99723697 evidence cleaner0 2023-07-26T11:53:36Z DUMMY: Structure 0.998892 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent RESULTS paragraph 7310 The mouse SEL1Lcent crystallized as a homodimer, and there were two homodimers in the crystal asymmetric unit (Fig. 1B,C, Supplementary Fig. 1). The two SEL1Lcent molecules dimerize in a head-to-tail manner through a two-fold symmetry interface resulting in a cosmos-like shaped structure (Fig. 1B). The resulting structure resembles the yin-yang symbol with overall dimensions of 60 × 60 × 25 Å, where a SEL1Lcent monomer corresponds to half the symbol. The dimer formation buries a surface area of 1670 Å2 for each monomer, and no significant differences between the protomers were displayed (final root mean square deviation (RMSD) of 0.6 Å for all Cα atoms). Each protomer is composed of ten α-helices, which form the five SLRs, resulting in an elongated curved structure, confirming the primary structure prediction (Fig. 1D). 0.99545884 taxonomy_domain cleaner0 2023-07-26T08:29:39Z DUMMY: mouse 0.99923635 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.9950051 experimental_method cleaner0 2023-07-26T12:14:55Z MESH: crystallized 0.9987419 oligomeric_state cleaner0 2023-07-26T08:24:28Z DUMMY: homodimer 0.9987446 oligomeric_state cleaner0 2023-07-26T08:39:18Z DUMMY: homodimers 0.9991404 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.9984725 oligomeric_state cleaner0 2023-07-26T08:38:55Z DUMMY: dimerize 0.9923738 protein_state cleaner0 2023-07-26T08:24:20Z DUMMY: head-to-tail 0.9871208 site cleaner0 2023-07-26T12:03:32Z SO: two-fold symmetry interface 0.89937943 evidence cleaner0 2023-07-26T11:54:22Z DUMMY: structure 0.99790156 evidence cleaner0 2023-07-26T11:54:25Z DUMMY: structure 0.9990891 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.9988864 oligomeric_state cleaner0 2023-07-26T08:39:01Z DUMMY: monomer 0.99883777 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.99886155 oligomeric_state cleaner0 2023-07-26T08:39:02Z DUMMY: monomer 0.99703 oligomeric_state cleaner0 2023-07-26T08:39:12Z DUMMY: protomers 0.99834615 evidence cleaner0 2023-07-26T11:54:29Z DUMMY: root mean square deviation 0.9983158 evidence cleaner0 2023-07-26T08:41:28Z DUMMY: RMSD 0.99887854 oligomeric_state cleaner0 2023-07-26T08:39:06Z DUMMY: protomer 0.998384 structure_element cleaner0 2023-07-26T09:48:10Z SO: α-helices 0.9965168 structure_element cleaner0 2023-07-26T08:41:13Z SO: SLRs RESULTS paragraph 8163 The α-helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs and SLRs. Helices A and B are 14 and 13 residues long, respectively, and the two helices are connected by a short turn and loop (Fig. 1D). In addition, a longer loop, consisting of approximately eight amino acids, is inserted between helix B of one SLR and helix A of the next SLR. This arrangement is a unique feature for SLRs among the major classes of repeats containing an α-solenoid. Starting from its N-terminus, the α-solenoid of SEL1L extends across a semi-circle in a right-handed superhelix fashion along the rotation axis of the yin-yang circle. However, the last helix, 9B, at the C-terminus adopts a different conformation, lying parallel to the long axis of helix 9A instead of forming an antiparallel SLR. This unique conformation of helix 9B most likely contributes to formation of the dimer structure of SEL1Lcent, as detailed below. With the exception of the last SLR, the four α-helix pairs possess similar conformations, with RMSD values of 0.7 Å for all Cα atoms. Although the sequence similarity for the pairwise alignments varies between 25% and 35%, all the residues present in the SLR motifs are conserved among the five pairs. The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525–533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible. 0.9993 structure_element cleaner0 2023-07-26T09:48:18Z SO: α-helices 0.8356808 evidence cleaner0 2023-07-26T11:54:34Z DUMMY: structure 0.99865365 structure_element cleaner0 2023-07-26T09:48:21Z SO: A 0.99602926 structure_element cleaner0 2023-07-26T09:48:24Z SO: B 0.99529266 structure_element cleaner0 2023-07-26T08:40:43Z SO: TPRs 0.93863434 structure_element cleaner0 2023-07-26T08:41:12Z SO: SLRs 0.9174499 structure_element cleaner0 2023-07-26T08:40:37Z SO: Helices A and B 0.964784 structure_element cleaner0 2023-07-26T09:48:28Z SO: helices 0.9637494 structure_element cleaner0 2023-07-26T09:48:34Z SO: turn 0.9960223 structure_element cleaner0 2023-07-26T09:48:36Z SO: loop 0.9986534 structure_element cleaner0 2023-07-26T09:48:39Z SO: loop 0.98835695 structure_element cleaner0 2023-07-26T08:40:26Z SO: helix B 0.99341077 structure_element cleaner0 2023-07-26T08:31:36Z SO: SLR 0.99514014 structure_element cleaner0 2023-07-26T08:40:32Z SO: helix A 0.9932869 structure_element cleaner0 2023-07-26T08:31:36Z SO: SLR 0.9989489 structure_element cleaner0 2023-07-26T08:41:13Z SO: SLRs 0.99916095 structure_element cleaner0 2023-07-26T09:48:43Z SO: α-solenoid 0.9992831 structure_element cleaner0 2023-07-26T09:48:46Z SO: α-solenoid 0.99934524 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.86892396 structure_element cleaner0 2023-07-26T08:40:53Z SO: yin-yang circle 0.9986014 structure_element cleaner0 2023-07-26T08:41:59Z SO: 9B structure_element SO: cleaner0 2023-07-26T08:40:19Z helix 9A 0.8358868 structure_element cleaner0 2023-07-26T08:31:36Z SO: SLR structure_element SO: cleaner0 2023-07-26T08:41:50Z helix 9B 0.99878865 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9993019 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.92849016 structure_element cleaner0 2023-07-26T08:31:36Z SO: SLR 0.99918014 structure_element cleaner0 2023-07-26T09:48:59Z SO: α-helix 0.9987 evidence cleaner0 2023-07-26T08:41:27Z DUMMY: RMSD 0.99687785 experimental_method cleaner0 2023-07-26T12:15:12Z MESH: pairwise alignments structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9889858 protein_state cleaner0 2023-07-26T08:42:36Z DUMMY: conserved structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR structure_element SO: cleaner0 2023-07-26T08:35:41Z SLR-M 0.9716945 residue_number cleaner0 2023-07-26T08:42:28Z DUMMY: 524 0.9968249 residue_range cleaner0 2023-07-26T08:42:24Z DUMMY: 525–533 0.99862355 evidence cleaner0 2023-07-26T08:42:46Z DUMMY: electron density map 0.99881995 protein_state cleaner0 2023-07-26T08:42:41Z DUMMY: highly flexible RESULTS paragraph 9617 Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1Lcent dimer shown in our crystal structure is a biological unit. First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. We detected bands at the mass of a dimer for both SEL1Lcent and SEL1Llong when cross-linked with low concentrations of GA (0.005%) or DMS (0.3 mM) (Supplementary Fig. 2A,B). Next, we conducted analytical ultracentrifugation of SEL1Lcent. Consistent with the cross-linking data, analytical ultracentrifugation revealed that the molecular weight of SEL1Lcent corresponds to a dimer (Supplementary Fig. 2C). Taken together, these data indicate that some kind of dimer is formed in solution. 0.9988261 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9988477 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9933989 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.99881846 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.99609435 evidence cleaner0 2023-07-26T11:52:55Z DUMMY: crystal structure 0.9954019 experimental_method cleaner0 2023-07-26T12:15:19Z MESH: cross-linked 0.99408615 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.9866093 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.99746454 mutant cleaner0 2023-07-26T08:43:07Z MESH: SEL1Llong 0.99769783 residue_range cleaner0 2023-07-26T08:43:15Z DUMMY: 337–554 0.9990128 chemical cleaner0 2023-07-26T08:43:22Z CHEBI: glutaraldehyde 0.9991844 chemical cleaner0 2023-07-26T08:43:26Z CHEBI: GA 0.9989731 chemical cleaner0 2023-07-26T08:43:31Z CHEBI: dimethyl suberimidate 0.99916816 chemical cleaner0 2023-07-26T08:43:36Z CHEBI: DMS 0.9988912 experimental_method cleaner0 2023-07-26T12:15:46Z MESH: SDS-PAGE 0.998814 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.99484646 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.9665328 mutant cleaner0 2023-07-26T08:43:07Z MESH: SEL1Llong 0.99625254 experimental_method cleaner0 2023-07-26T12:15:28Z MESH: cross-linked 0.9990452 chemical cleaner0 2023-07-26T08:43:27Z CHEBI: GA 0.9989182 chemical cleaner0 2023-07-26T08:43:37Z CHEBI: DMS 0.9989053 experimental_method cleaner0 2023-07-26T12:15:53Z MESH: analytical ultracentrifugation 0.99510103 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.99864703 experimental_method cleaner0 2023-07-26T12:16:01Z MESH: cross-linking 0.9988791 experimental_method cleaner0 2023-07-26T12:16:06Z MESH: analytical ultracentrifugation evidence DUMMY: cleaner0 2023-07-26T12:16:18Z molecular weight 0.9970095 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.99879503 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.99886096 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer RESULTS title_2 10509 Dimer Interface of SEL1Lcent 0.99903053 site cleaner0 2023-07-26T08:43:53Z SO: Dimer Interface 0.99924254 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent RESULTS paragraph 10538 In contrast to a previously described SLR motif containing proteins that exist as monomers in solution, SEL1Lcent forms an intimate two-fold homotypic dimer interface (Figs 1B and 2A). The concave surface of each SEL1L domain comprising helix 5A to 9A encircles its dimer counterpart in an interlocking clasp-like arrangement. However, no interactions were seen between the two-fold-related protomers through the concave inner surfaces themselves. Rather, the unique structure of SLR motif 9, consisting of two parallel helices (9A and 9B), is located in the space generated by the concave surface and provides an extensive dimerization interface between the two-fold-related molecules (Fig. 2A). Helix 9B from one protomer inserts into the empty space surrounded by the concave region in the other monomer, forming a domain-swapped conformation. protein_type MESH: cleaner0 2023-07-26T08:45:41Z SLR motif containing proteins 0.99890316 oligomeric_state cleaner0 2023-07-26T11:55:54Z DUMMY: monomers 0.998408 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent site SO: cleaner0 2023-07-26T08:46:09Z two-fold homotypic dimer interface 0.98965824 site cleaner0 2023-07-26T08:46:22Z SO: concave surface 0.9853652 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:44:27Z helix 5A to 9A 0.9988918 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9983084 oligomeric_state cleaner0 2023-07-26T08:39:13Z DUMMY: protomers 0.9283633 site cleaner0 2023-07-26T12:03:37Z SO: concave inner surfaces 0.99800134 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.9989574 structure_element cleaner0 2023-07-26T08:44:43Z SO: 9A 0.99379176 structure_element cleaner0 2023-07-26T08:44:45Z SO: 9B 0.9734504 site cleaner0 2023-07-26T08:46:23Z SO: concave surface 0.9988966 site cleaner0 2023-07-26T08:58:10Z SO: dimerization interface structure_element SO: cleaner0 2023-07-26T08:41:50Z Helix 9B 0.99881697 oligomeric_state cleaner0 2023-07-26T08:39:07Z DUMMY: protomer 0.963019 site cleaner0 2023-07-26T12:03:42Z SO: concave region 0.99887115 oligomeric_state cleaner0 2023-07-26T08:39:02Z DUMMY: monomer protein_state DUMMY: cleaner0 2023-07-26T09:03:04Z domain-swapped RESULTS paragraph 11385 Three major contact interfaces are involved in the interactions, and all interfaces are symmetrically related between the dimer subunits (Fig. 2A). Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs (Fig. 1E). First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. The side chain of Gln 523 forms an H-bond to the side chain of Asp 480 on the two-fold-related protomer (Fig. 2A, Interface 1 detail). Second, the residues from helix 9A interact with the residues from helix 5A of its counterpart in a head-to-tail orientation. In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). Third, the helix 9B from each protomer is involved in the dimer interaction by forming a two-fold antiparallel symmetry. In particular, the side chains of hydrophobic residues, including Phe 518, Leu 521, and Met 524, are directed toward each other, where they make both inter- and intramolecular contacts (Fig. 2A, Interface 3 detail). 0.998649 site cleaner0 2023-07-26T12:03:48Z SO: contact interfaces 0.9989027 site cleaner0 2023-07-26T12:03:55Z SO: interfaces 0.9985661 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9989074 experimental_method cleaner0 2023-07-26T12:16:44Z MESH: Structure-based sequence alignment 0.9986141 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.76681197 experimental_method cleaner0 2023-07-26T12:16:49Z MESH: ConSurf server 0.9986466 site cleaner0 2023-07-26T08:47:05Z SO: dimer interfaces 0.9988012 protein_state cleaner0 2023-07-26T08:47:52Z DUMMY: highly conserved 0.921708 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.9958381 structure_element cleaner0 2023-07-26T08:41:50Z SO: helix 9B 0.9777349 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent 0.9805442 site cleaner0 2023-07-26T12:04:23Z SO: inner groove 0.99077106 structure_element cleaner0 2023-07-26T08:31:36Z SO: SLR 0.9991037 structure_element cleaner0 2023-07-26T08:47:40Z SO: α-helices 0.9984108 structure_element cleaner0 2023-07-26T08:47:43Z SO: 5B 0.9963392 structure_element cleaner0 2023-07-26T08:47:45Z SO: 6B 0.9923379 structure_element cleaner0 2023-07-26T08:47:48Z SO: 7B 0.99450463 structure_element cleaner0 2023-07-26T08:47:50Z SO: 8B 0.99847454 site cleaner0 2023-07-26T12:04:30Z SO: interface 0.9957429 residue_name_number cleaner0 2023-07-26T08:48:11Z DUMMY: Leu 516 0.9974791 residue_name_number cleaner0 2023-07-26T08:48:16Z DUMMY: Tyr 519 0.9773166 structure_element cleaner0 2023-07-26T08:41:50Z SO: helix 9B 0.9973022 bond_interaction cleaner0 2023-07-26T08:50:40Z MESH: hydrophobic interactions 0.9968979 residue_name_number cleaner0 2023-07-26T08:48:22Z DUMMY: Trp 478 0.97454387 structure_element cleaner0 2023-07-26T08:46:46Z SO: helix 8B 0.99696076 residue_name_number cleaner0 2023-07-26T08:48:26Z DUMMY: Val 444 0.9958078 structure_element cleaner0 2023-07-26T08:46:51Z SO: helix 7B 0.9978001 residue_name_number cleaner0 2023-07-26T08:48:31Z DUMMY: Phe 411 0.9964856 structure_element cleaner0 2023-07-26T08:47:58Z SO: helix 6B 0.99774224 residue_name_number cleaner0 2023-07-26T08:48:39Z DUMMY: Leu 380 0.9722063 structure_element cleaner0 2023-07-26T08:48:03Z SO: helix 5B 0.9914647 structure_element cleaner0 2023-07-26T08:23:45Z SO: SEL1Lcent site SO: cleaner0 2023-07-26T08:51:38Z Interface 1 0.9971962 bond_interaction cleaner0 2023-07-26T08:50:40Z MESH: hydrophobic interactions 0.9972205 residue_name_number cleaner0 2023-07-26T08:48:16Z DUMMY: Tyr 519 0.9973451 residue_name_number cleaner0 2023-07-26T08:48:47Z DUMMY: Ile 515 0.99737597 bond_interaction cleaner0 2023-07-26T08:50:46Z MESH: H-bonds 0.9988205 protein_state cleaner0 2023-07-26T12:00:49Z DUMMY: conserved 0.9975953 residue_name_number cleaner0 2023-07-26T08:48:54Z DUMMY: Gln 377 0.99773026 residue_name_number cleaner0 2023-07-26T08:48:59Z DUMMY: His 381 0.9872621 structure_element cleaner0 2023-07-26T08:48:03Z SO: helix 5B 0.9984987 oligomeric_state cleaner0 2023-07-26T08:39:07Z DUMMY: protomer 0.9967507 residue_name_number cleaner0 2023-07-26T08:49:04Z DUMMY: Gln 523 0.99719113 bond_interaction cleaner0 2023-07-26T08:50:51Z MESH: H-bond 0.99751633 residue_name_number cleaner0 2023-07-26T08:49:10Z DUMMY: Asp 480 0.99792105 oligomeric_state cleaner0 2023-07-26T08:39:07Z DUMMY: protomer site SO: cleaner0 2023-07-26T08:51:38Z Interface 1 0.9912703 structure_element cleaner0 2023-07-26T08:40:20Z SO: helix 9A 0.99822116 structure_element cleaner0 2023-07-26T09:49:09Z SO: helix 5A 0.9975549 protein_state cleaner0 2023-07-26T08:24:20Z DUMMY: head-to-tail 0.99807405 site cleaner0 2023-07-26T12:04:35Z SO: interface 0.99662375 structure_element cleaner0 2023-07-26T08:40:20Z SO: helix 9A 0.99731815 residue_name_number cleaner0 2023-07-26T08:49:17Z DUMMY: Leu 503 0.9975286 residue_name_number cleaner0 2023-07-26T08:49:21Z DUMMY: Tyr 499 0.99735534 residue_name_number cleaner0 2023-07-26T08:49:27Z DUMMY: Lys 500 0.993017 bond_interaction cleaner0 2023-07-26T08:50:55Z MESH: van der Waals contacts 0.99887514 structure_element cleaner0 2023-07-26T09:49:09Z SO: helix 5A 0.9965479 residue_name_number cleaner0 2023-07-26T08:49:32Z DUMMY: Tyr 360 0.9968129 residue_name_number cleaner0 2023-07-26T08:49:37Z DUMMY: Leu 356 0.99606204 residue_name_number cleaner0 2023-07-26T08:49:42Z DUMMY: Tyr 359 0.99658686 residue_name_number cleaner0 2023-07-26T08:49:46Z DUMMY: Leu 363 0.9970648 bond_interaction cleaner0 2023-07-26T08:50:40Z MESH: hydrophobic interactions 0.99649847 residue_name_number cleaner0 2023-07-26T08:49:51Z DUMMY: Asn 507 0.9968495 residue_name_number cleaner0 2023-07-26T08:49:56Z DUMMY: Ser 510 0.98241115 structure_element cleaner0 2023-07-26T08:40:20Z SO: helix 9A 0.9971602 bond_interaction cleaner0 2023-07-26T08:50:47Z MESH: H-bonds 0.99891543 protein_state cleaner0 2023-07-26T08:50:34Z DUMMY: highly conserved 0.9974524 residue_name_number cleaner0 2023-07-26T08:50:02Z DUMMY: Arg 384 0.9959947 structure_element cleaner0 2023-07-26T09:49:14Z SO: loop 0.99705195 structure_element cleaner0 2023-07-26T08:48:03Z SO: helix 5B 0.9963291 structure_element cleaner0 2023-07-26T08:50:20Z SO: 6A 0.99503314 oligomeric_state cleaner0 2023-07-26T08:39:07Z DUMMY: protomer 0.99800134 structure_element cleaner0 2023-07-26T08:41:50Z SO: helix 9B 0.99873155 oligomeric_state cleaner0 2023-07-26T08:39:07Z DUMMY: protomer 0.99886876 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.996307 residue_name_number cleaner0 2023-07-26T08:50:06Z DUMMY: Phe 518 0.9968066 residue_name_number cleaner0 2023-07-26T08:50:10Z DUMMY: Leu 521 0.9973854 residue_name_number cleaner0 2023-07-26T08:50:15Z DUMMY: Met 524 site SO: cleaner0 2023-07-26T08:53:07Z Interface 3 RESULTS paragraph 13524 To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). However, the mutant behaved as a monomer in size-exclusion chromatography and analytical ultracentrifugation experiments (Fig. 2B, Supplementary Fig. 2C). Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type. Notably, a single-residue mutation (L521A in interface 3) abolished the dimerization of SEL1Lcent (Fig. 2B). Leu 521 is located in the dimerization center of the antiparallel 9B helices in the SEL1Lcent dimer. 0.99796003 evidence cleaner0 2023-07-26T11:52:55Z DUMMY: crystal structure 0.99194884 protein_state cleaner0 2023-07-26T08:53:27Z DUMMY: deletion mutant mutant MESH: cleaner0 2023-07-26T09:04:56Z SEL1L348–497 0.99806386 protein_state cleaner0 2023-07-26T12:00:55Z DUMMY: lacking 0.9974417 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 structure_element SO: cleaner0 2023-07-26T08:40:20Z helix 9A 0.99706894 structure_element cleaner0 2023-07-26T08:51:24Z SO: 9B 0.9993037 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.97612184 protein_state cleaner0 2023-07-26T08:53:26Z DUMMY: deletion mutant 0.9989631 protein_state cleaner0 2023-07-26T08:53:20Z DUMMY: wild-type 0.9991844 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent evidence DUMMY: cleaner0 2023-07-26T08:53:45Z spectra 0.9971682 experimental_method cleaner0 2023-07-26T12:16:53Z MESH: CD spectroscopy 0.9705612 experimental_method cleaner0 2023-07-26T12:16:58Z MESH: deletion 0.9956174 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.99916804 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.9989379 protein_state cleaner0 2023-07-26T12:00:59Z DUMMY: mutant 0.9988734 oligomeric_state cleaner0 2023-07-26T08:39:02Z DUMMY: monomer 0.9987341 experimental_method cleaner0 2023-07-26T12:17:01Z MESH: size-exclusion chromatography 0.9985672 experimental_method cleaner0 2023-07-26T12:17:04Z MESH: analytical ultracentrifugation 0.9988134 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer protein_state DUMMY: cleaner0 2023-07-26T08:52:00Z triple point mutant 0.99679816 site cleaner0 2023-07-26T08:51:37Z SO: Interface 1 0.9986395 mutant cleaner0 2023-07-26T08:52:10Z MESH: I515A 0.99888664 mutant cleaner0 2023-07-26T08:52:14Z MESH: L516A 0.998955 mutant cleaner0 2023-07-26T08:52:19Z MESH: Y519A oligomeric_state DUMMY: cleaner0 2023-07-26T11:57:09Z dimerization 0.78520614 protein_state cleaner0 2023-07-26T08:52:01Z DUMMY: triple point mutant 0.9985977 oligomeric_state cleaner0 2023-07-26T08:39:02Z DUMMY: monomer 0.99828404 experimental_method cleaner0 2023-07-26T12:17:07Z MESH: size-exclusion chromatography 0.7539347 protein_state cleaner0 2023-07-26T08:52:56Z DUMMY: point mutant 0.9990096 mutant cleaner0 2023-07-26T08:52:53Z MESH: Q460A 0.9989252 protein_state cleaner0 2023-07-26T08:53:21Z DUMMY: wild-type experimental_method MESH: cleaner0 2023-07-26T08:52:45Z single-residue mutation 0.99892384 mutant cleaner0 2023-07-26T08:52:49Z MESH: L521A 0.9969276 site cleaner0 2023-07-26T08:53:06Z SO: interface 3 protein_state DUMMY: cleaner0 2023-07-26T08:54:10Z abolished the dimerization 0.99925107 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.9963031 residue_name_number cleaner0 2023-07-26T08:50:11Z DUMMY: Leu 521 0.99869436 site cleaner0 2023-07-26T11:58:04Z SO: dimerization center 0.9960935 structure_element cleaner0 2023-07-26T09:49:23Z SO: 9B helices 0.9992654 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.998847 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer RESULTS paragraph 14728 Taken together, these structural and biochemical data demonstrate that SEL1Lcent exists as a dimer in solution and that SLR motif 9 in SEL1Lcent plays an important role in generating a two-fold dimerization interface. 0.99832624 evidence cleaner0 2023-07-26T11:54:41Z DUMMY: structural and biochemical data 0.9992519 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.99875975 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.99915147 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.9993118 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.99687177 site cleaner0 2023-07-26T08:58:10Z SO: dimerization interface RESULTS title_2 14946 The Two Glycine Residues (G512 and G513) Create a Hinge for Domain Swapping of SLR Motif 9 0.9976674 residue_name cleaner0 2023-07-26T12:27:43Z SO: Glycine 0.9995813 residue_name_number cleaner0 2023-07-26T08:58:15Z DUMMY: G512 0.9995747 residue_name_number cleaner0 2023-07-26T08:58:21Z DUMMY: G513 0.9722741 structure_element cleaner0 2023-07-26T09:00:42Z SO: Hinge structure_element SO: cleaner0 2023-07-26T08:25:42Z SLR Motif 9 RESULTS paragraph 15037 SLRs of mouse SEL1L were predicted using the TPRpred server. Based on the prediction, full-length SEL1L contains a total of 11 SLR motifs, and our construct corresponds to SLR motifs 5 through 9. Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif. According to our crystal structure, the central axis of helix 9B is almost parallel to that of helix 9A (Fig. 3B). However, this unusual conformation of SLR motif 9 seems to be essential for dimer formation, as described earlier. For this structural geometry, two adjacent residues, Gly 512 and Gly 513, in SEL1L confer flexibility at this position by adopting main-chain dihedral angles that are disallowed for non-glycine residues. The phi and psi dihedrals are 100° and 20° for Gly 512, and 110° and −20° for Gly 513, respectively (Fig. 3C). Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A). Thus, the Gly-Gly residues generate an unusual sharp bend at the C-terminal SLR motif 9. The involvement of a glycine residue in forming a hinge for domain swapping has been reported previously. The significance of Gly 513 is further highlighted by its absolute conservation among different species, including the budding yeast homolog Hrd3p. 0.99879164 structure_element cleaner0 2023-07-26T08:41:13Z SO: SLRs 0.99712 taxonomy_domain cleaner0 2023-07-26T08:29:39Z DUMMY: mouse 0.9992617 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.99805737 experimental_method cleaner0 2023-07-26T12:17:13Z MESH: TPRpred server 0.99909896 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.9992016 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9956316 structure_element cleaner0 2023-07-26T09:49:28Z SO: SLR motifs 5 through 9 structure_element SO: cleaner0 2023-07-26T08:40:20Z helix 9A 0.9945779 structure_element cleaner0 2023-07-26T08:59:33Z SO: 9B 0.9980234 structure_element cleaner0 2023-07-26T08:59:40Z SO: SLR repeats 0.9980931 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.9152161 structure_element cleaner0 2023-07-26T09:49:33Z SO: helices structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9985614 evidence cleaner0 2023-07-26T11:52:55Z DUMMY: crystal structure structure_element SO: cleaner0 2023-07-26T08:41:50Z helix 9B structure_element SO: cleaner0 2023-07-26T08:40:20Z helix 9A 0.9983694 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.99886876 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9963238 residue_name_number cleaner0 2023-07-26T09:34:15Z DUMMY: Gly 512 0.9969952 residue_name_number cleaner0 2023-07-26T09:34:18Z DUMMY: Gly 513 0.99919623 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.99500006 residue_name_number cleaner0 2023-07-26T09:34:15Z DUMMY: Gly 512 0.9959071 residue_name_number cleaner0 2023-07-26T09:34:18Z DUMMY: Gly 513 0.9964285 residue_name_number cleaner0 2023-07-26T09:34:18Z DUMMY: Gly 513 0.9982552 protein_state cleaner0 2023-07-26T09:00:04Z DUMMY: conserved structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.99914706 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.99679244 residue_name_number cleaner0 2023-07-26T09:34:15Z DUMMY: Gly 512 0.9982619 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.9991812 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent structure_element SO: cleaner0 2023-07-26T09:01:23Z Gly-Gly 0.99818 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.9979327 residue_name cleaner0 2023-07-26T09:00:37Z SO: glycine 0.9945412 structure_element cleaner0 2023-07-26T09:00:43Z SO: hinge 0.9972619 residue_name_number cleaner0 2023-07-26T09:34:18Z DUMMY: Gly 513 0.98326504 protein_state cleaner0 2023-07-26T09:01:33Z DUMMY: absolute conservation 0.9730308 taxonomy_domain cleaner0 2023-07-26T09:00:54Z DUMMY: budding yeast 0.99933845 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p RESULTS paragraph 16501 To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). This means that the effect of the mutation is mainly to generate a more restricted geometry at the hinge region. G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B. However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. The G512K/G513K double mutant eluted at the monomer position in size-exclusion chromatography (Fig. 3D). A previous study shows that induction of steric hindrance by mutation destabilizes the dimerization interface of a different protein, ClC transporter. 0.9956256 residue_name_number cleaner0 2023-07-26T09:34:15Z DUMMY: Gly 512 0.9962336 residue_name_number cleaner0 2023-07-26T09:34:18Z DUMMY: Gly 513 structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9958221 experimental_method cleaner0 2023-07-26T12:17:27Z MESH: point mutation 0.8371418 mutant cleaner0 2023-07-26T09:01:49Z MESH: Gly to Ala 0.99523824 residue_name_number cleaner0 2023-07-26T09:34:15Z DUMMY: Gly 512 0.9954579 residue_name_number cleaner0 2023-07-26T09:34:19Z DUMMY: Gly 513 0.9959911 structure_element cleaner0 2023-07-26T08:41:50Z SO: helix 9B 0.994381 oligomeric_state cleaner0 2023-07-26T08:39:07Z DUMMY: protomer 0.9974095 residue_name cleaner0 2023-07-26T09:01:59Z SO: alanine 0.9810831 experimental_method cleaner0 2023-07-26T12:17:36Z MESH: mutation 0.9816042 experimental_method cleaner0 2023-07-26T12:17:38Z MESH: mutation structure_element SO: cleaner0 2023-07-26T09:00:43Z hinge 0.9990533 mutant cleaner0 2023-07-26T09:02:13Z MESH: G512A 0.99901223 mutant cleaner0 2023-07-26T09:02:17Z MESH: G513A 0.9987969 protein_state cleaner0 2023-07-26T08:53:21Z DUMMY: wild-type 0.99452174 experimental_method cleaner0 2023-07-26T12:17:41Z MESH: size-exclusion chromatography 0.9920854 residue_name cleaner0 2023-07-26T09:02:26Z SO: glycine 0.99700415 structure_element cleaner0 2023-07-26T08:41:50Z SO: helix 9B 0.89237595 protein_state cleaner0 2023-07-26T09:02:33Z DUMMY: double mutant 0.9990127 mutant cleaner0 2023-07-26T09:02:13Z MESH: G512A 0.9969656 mutant cleaner0 2023-07-26T09:02:18Z MESH: G513A 0.998868 protein_state cleaner0 2023-07-26T08:53:21Z DUMMY: wild-type 0.9976515 experimental_method cleaner0 2023-07-26T12:17:44Z MESH: mutation 0.9992169 structure_element cleaner0 2023-07-26T09:00:43Z SO: hinge 0.9940084 structure_element cleaner0 2023-07-26T08:40:20Z SO: helix 9A 0.89214116 structure_element cleaner0 2023-07-26T09:49:44Z SO: 9B 0.9929811 structure_element cleaner0 2023-07-26T08:41:50Z SO: helix 9B 0.9993969 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.98809755 experimental_method cleaner0 2023-07-26T12:17:51Z MESH: mutated to 0.99598 residue_name cleaner0 2023-07-26T09:02:28Z SO: lysine 0.9989839 mutant cleaner0 2023-07-26T09:02:42Z MESH: G512K 0.99544466 mutant cleaner0 2023-07-26T09:02:47Z MESH: G513K 0.9972485 protein_state cleaner0 2023-07-26T09:02:37Z DUMMY: mutant 0.9987363 structure_element cleaner0 2023-07-26T09:00:43Z SO: hinge 0.9963349 oligomeric_state cleaner0 2023-07-26T08:39:07Z DUMMY: protomer 0.99938715 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.9994 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.9989249 mutant cleaner0 2023-07-26T09:02:43Z MESH: G512K 0.99053127 mutant cleaner0 2023-07-26T09:02:47Z MESH: G513K 0.93179905 protein_state cleaner0 2023-07-26T09:02:33Z DUMMY: double mutant 0.9976248 oligomeric_state cleaner0 2023-07-26T08:39:02Z DUMMY: monomer 0.9979068 experimental_method cleaner0 2023-07-26T12:17:55Z MESH: size-exclusion chromatography 0.5608726 experimental_method cleaner0 2023-07-26T12:17:59Z MESH: mutation 0.9989898 site cleaner0 2023-07-26T08:58:10Z SO: dimerization interface 0.98787004 protein_type cleaner0 2023-07-26T12:18:03Z MESH: ClC transporter RESULTS paragraph 18214 Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation. 0.9961103 residue_name_number cleaner0 2023-07-26T09:34:15Z DUMMY: Gly 512 0.9966997 residue_name_number cleaner0 2023-07-26T09:34:19Z DUMMY: Gly 513 0.99120015 structure_element cleaner0 2023-07-26T08:40:20Z SO: helix 9A 0.5932326 structure_element cleaner0 2023-07-26T09:49:50Z SO: 9B 0.99841547 protein_state cleaner0 2023-07-26T09:03:04Z DUMMY: domain-swapped 0.9987753 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer RESULTS title_2 18411 SEL1L Forms Self-oligomers through SEL1Lcent domain in vivo 0.9990978 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L oligomeric_state DUMMY: cleaner0 2023-07-26T09:06:04Z Self-oligomers 0.99933666 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent RESULTS paragraph 18471 Next, we examined if SEL1L also forms self-oligomers in vivo using HEK293T cells. We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. Co-immunoprecipitation analysis showed that the SEL1Lcent was sufficient to physically interact with the full-length SEL1L, while SEL1L348–497 failed to do so (Fig. 4A). Interestingly, however, the expression level of SEL1L348–497 was consistently lower than that of SEL1Lcent (Fig. 4A,B). Semi-quantitative RT-PCR revealed no significant difference in transcriptional levels of the two constructs (data not shown). We speculated that SEL1L348–497 could be secreted while the SEL1Lcent is retained in the ER by association with the endogenous ERAD complex. Indeed, immunoprecipitation followed by western blot analysis using the culture medium detected secreted SEL1L348–497 fragment, but not SEL1Lcent (Fig. 4B). We next examined if the reason why SEL1L348–497 failed to bind to the full-length SEL1L may be because of the lower level of SEL1L348–497 in the ER lumen compared to SEL1Lcent fragment. In order to retain two SEL1L fragments in the ER lumen, we added KDEL ER retention sequence to the C-terminus of both fragments. Indeed, the addition of KDEL peptide increased the level of SEL1L348–497 in the ER lumen (Fig. 4D,E) and the immunostaining analysis showed both constructs were well localized to the ER (Fig. 4C). We further analyzed whether SEL1Lcent may competitively inhibit the self-oligomerization of SEL1L in vivo. To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. Co-immunoprecipitation assay revealed that wild-type SEL1Lcent-KDEL, indeed, competitively disrupted the self-association of the full-length SEL1L (Fig. 4E). In contrast, SEL1L348–497-KDEL and the single-residue mutation L521A in SEL1Lcent did not competitively inhibit the self-association of full-length SEL1L (Fig. 4E,F). These data suggest that the SEL1L forms self-oligomers and the oligomerization is mediated by the SEL1Lcent domain in vivo. 0.99905807 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L oligomeric_state DUMMY: cleaner0 2023-07-26T09:06:04Z self-oligomers 0.9990368 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.99192 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L experimental_method MESH: cleaner0 2023-07-26T09:03:35Z HA 0.9596383 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.60219574 experimental_method cleaner0 2023-07-26T09:03:44Z MESH: FLAG experimental_method MESH: cleaner0 2023-07-26T12:18:38Z fusion constructs 0.9982791 experimental_method cleaner0 2023-07-26T12:18:41Z MESH: co-transfected 0.9990059 experimental_method cleaner0 2023-07-26T12:18:44Z MESH: co-immunoprecipitation assay experimental_method MESH: cleaner0 2023-07-26T09:03:45Z FLAG 0.9982916 experimental_method cleaner0 2023-07-26T12:18:49Z MESH: Western blot experimental_method MESH: cleaner0 2023-07-26T09:09:28Z HA 0.999084 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.9989919 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L oligomeric_state DUMMY: cleaner0 2023-07-26T09:06:04Z self-oligomers 0.8052421 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.9991011 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.9988022 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.8979191 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent structure_element SO: cleaner0 2023-07-26T09:04:35Z SLR motif 9 experimental_method MESH: cleaner0 2023-07-26T09:04:44Z deletion 0.79511404 mutant cleaner0 2023-07-26T09:04:55Z MESH: SEL1L348–497 0.88660175 experimental_method cleaner0 2023-07-26T12:18:55Z MESH: fused to 0.9970625 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.9947206 structure_element cleaner0 2023-07-26T09:05:43Z SO: signal peptides 0.9989898 experimental_method cleaner0 2023-07-26T12:18:58Z MESH: Co-immunoprecipitation analysis 0.6223402 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.9991303 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.9983606 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.8710802 mutant cleaner0 2023-07-26T09:04:56Z MESH: SEL1L348–497 0.8748493 mutant cleaner0 2023-07-26T09:04:56Z MESH: SEL1L348–497 0.5263394 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.9990075 experimental_method cleaner0 2023-07-26T12:19:05Z MESH: Semi-quantitative RT-PCR mutant MESH: cleaner0 2023-07-26T09:04:56Z SEL1L348–497 0.91881275 structure_element cleaner0 2023-07-26T08:23:46Z SO: SEL1Lcent 0.9988921 experimental_method cleaner0 2023-07-26T12:19:09Z MESH: immunoprecipitation 0.9573611 experimental_method cleaner0 2023-07-26T12:19:12Z MESH: western blot mutant MESH: cleaner0 2023-07-26T09:04:56Z SEL1L348–497 0.9566656 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent 0.8454278 mutant cleaner0 2023-07-26T09:04:56Z MESH: SEL1L348–497 0.9990664 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.99819946 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L mutant MESH: cleaner0 2023-07-26T09:04:56Z SEL1L348–497 0.86057234 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent 0.87872255 protein cleaner0 2023-07-26T08:24:00Z PR: SEL1L 0.7766495 structure_element cleaner0 2023-07-26T12:19:53Z SO: KDEL 0.9066809 structure_element cleaner0 2023-07-26T09:50:01Z SO: ER retention sequence structure_element SO: cleaner0 2023-07-26T12:20:10Z KDEL mutant MESH: cleaner0 2023-07-26T09:04:56Z SEL1L348–497 0.9971819 experimental_method cleaner0 2023-07-26T12:19:16Z MESH: immunostaining 0.7080341 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent 0.99902713 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.99842024 experimental_method cleaner0 2023-07-26T12:19:20Z MESH: co-transfected 0.9777483 protein_state cleaner0 2023-07-26T12:01:08Z DUMMY: tagged 0.9990776 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.9985807 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.99638003 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.4470757 experimental_method cleaner0 2023-07-26T09:09:27Z MESH: HA 0.98915076 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L experimental_method MESH: cleaner0 2023-07-26T09:03:45Z FLAG 0.89780843 experimental_method cleaner0 2023-07-26T12:19:26Z MESH: increasing doses mutant MESH: cleaner0 2023-07-26T09:07:32Z SEL1Lcent-KDEL mutant MESH: cleaner0 2023-07-26T09:08:07Z SEL1L348–497-KDEL mutant MESH: cleaner0 2023-07-26T09:09:00Z SEL1Lcent (L521A)-KDEL 0.9989825 experimental_method cleaner0 2023-07-26T12:19:29Z MESH: Co-immunoprecipitation assay protein_state DUMMY: cleaner0 2023-07-26T08:53:21Z wild-type mutant MESH: cleaner0 2023-07-26T09:07:33Z SEL1Lcent-KDEL protein_state DUMMY: cleaner0 2023-07-26T08:24:14Z full-length mutant MESH: cleaner0 2023-07-26T09:08:08Z SEL1L348–497-KDEL mutant MESH: cleaner0 2023-07-26T08:52:49Z L521A structure_element SO: cleaner0 2023-07-26T08:23:47Z SEL1Lcent protein_state DUMMY: cleaner0 2023-07-26T08:24:14Z full-length protein PR: cleaner0 2023-07-26T08:24:01Z SEL1L protein PR: cleaner0 2023-07-26T08:24:01Z SEL1L oligomeric_state DUMMY: cleaner0 2023-07-26T09:06:04Z self-oligomers structure_element SO: cleaner0 2023-07-26T08:23:47Z SEL1Lcent RESULTS title_2 21109 Structural Comparison of SEL1L SLRs with TPRs or SLRs of Other Proteins 0.99825406 experimental_method cleaner0 2023-07-26T12:20:20Z MESH: Structural Comparison 0.99931073 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.99896467 structure_element cleaner0 2023-07-26T08:41:13Z SO: SLRs 0.99888355 structure_element cleaner0 2023-07-26T08:40:43Z SO: TPRs 0.99914634 structure_element cleaner0 2023-07-26T08:41:13Z SO: SLRs RESULTS paragraph 21181 Previous studies reveal that TPRs and SLRs have similar consensus sequences, suggesting that their three-dimensional structures are also similar. The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). The differences may result from the differing numbers of residues in the loops and differences in antiparallel helix packing. Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1Lcent. These factors contribute to the differences in the overall conformation of the SLR motifs in SEL1L and other SLR or TPR motif-containing proteins. 0.99915004 structure_element cleaner0 2023-07-26T08:40:43Z SO: TPRs 0.9991611 structure_element cleaner0 2023-07-26T08:41:13Z SO: SLRs 0.9988194 experimental_method cleaner0 2023-07-26T12:20:23Z MESH: superposition 0.9993436 structure_element cleaner0 2023-07-26T08:40:43Z SO: TPRs 0.9990426 protein cleaner0 2023-07-26T09:10:13Z PR: Cdc23 0.9972758 species cleaner0 2023-07-26T09:10:05Z MESH: S. pombe protein PR: cleaner0 2023-07-26T09:10:01Z cell division cycle 23 0.9991893 structure_element cleaner0 2023-07-26T08:41:13Z SO: SLRs 0.9991386 protein cleaner0 2023-07-26T09:10:22Z PR: HcpC 0.9924793 protein cleaner0 2023-07-26T09:10:29Z PR: Helicobacter Cysteine-rich Protein C 0.9976896 evidence cleaner0 2023-07-26T11:54:48Z DUMMY: RMSDs structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9987198 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9984101 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent 0.9481077 experimental_method cleaner0 2023-07-26T12:20:34Z MESH: structural alignment 0.99925023 structure_element cleaner0 2023-07-26T08:40:43Z SO: TPRs 0.99756396 evidence cleaner0 2023-07-26T08:41:28Z DUMMY: RMSD protein PR: cleaner0 2023-07-26T09:11:47Z Cdc23 0.9993161 structure_element cleaner0 2023-07-26T08:41:13Z SO: SLRs 0.9979068 evidence cleaner0 2023-07-26T08:41:28Z DUMMY: RMSD 0.99843234 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.9988809 experimental_method cleaner0 2023-07-26T12:20:37Z MESH: superimposing 0.9978993 evidence cleaner0 2023-07-26T11:54:52Z DUMMY: structure 0.96890366 structure_element cleaner0 2023-07-26T08:25:36Z SO: SLR motifs 5 to 9 0.9973239 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent protein PR: cleaner0 2023-07-26T09:12:02Z Cdc23 0.9991424 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.99682415 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.9979621 evidence cleaner0 2023-07-26T11:54:56Z DUMMY: structures 0.969845 structure_element cleaner0 2023-07-26T08:25:36Z SO: SLR motifs 5 to 9 0.9980794 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent 0.9989575 protein cleaner0 2023-07-26T09:10:14Z PR: Cdc23 0.9989826 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.99841034 evidence cleaner0 2023-07-26T08:41:28Z DUMMY: RMSD 0.9975805 structure_element cleaner0 2023-07-26T09:50:22Z SO: loops structure_element SO: cleaner0 2023-07-26T09:50:44Z antiparallel helix 0.99637115 protein_state cleaner0 2023-07-26T09:12:07Z DUMMY: conserved 0.98580664 ptm cleaner0 2023-07-26T09:12:10Z MESH: disulfide bonds structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9988392 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.99880195 protein cleaner0 2023-07-26T09:42:10Z PR: HcpB 0.99857056 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent structure_element SO: cleaner0 2023-07-26T08:31:36Z SLR 0.9981993 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L protein_type MESH: cleaner0 2023-07-26T09:12:43Z SLR or TPR motif-containing proteins RESULTS paragraph 22532 Another major difference in the structure of SLR motifs between SEL1L and HcpC is the oligomeric state of proteins. The TPR motif is involved in the dimerization of proteins such as Cdc23, Cdc16, and Cdc27. In particular, the N-terminal domain of Cdc23 (Cdc23N-term) has a TPR-motif organization similar to that of the SLR motif in SEL1Lcent. The seven TPR motifs of Cdc23N-term are assembled into a superhelical structure, generating a hollow surface and encircling its dimer counterpart in an interlocking clasp-like arrangement (Fig. 5C). The TPR motif 1 (TPR1) of each Cdc23N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR α-helices, generating two-fold symmetry homotype interactions. However, in this structure, a conformational change in the TPR motif itself is not observed. 0.99250495 evidence cleaner0 2023-07-26T09:44:28Z DUMMY: structure structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.99894506 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.99799466 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC structure_element SO: cleaner0 2023-07-26T09:43:43Z TPR oligomeric_state DUMMY: cleaner0 2023-07-26T11:57:09Z dimerization 0.99848664 protein cleaner0 2023-07-26T09:10:14Z PR: Cdc23 0.99822587 protein cleaner0 2023-07-26T09:13:06Z PR: Cdc16 0.9979845 protein cleaner0 2023-07-26T09:13:11Z PR: Cdc27 0.9977392 protein cleaner0 2023-07-26T09:10:14Z PR: Cdc23 protein PR: cleaner0 2023-07-26T09:13:28Z Cdc23 structure_element SO: cleaner0 2023-07-26T09:43:43Z TPR structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9988959 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent structure_element SO: cleaner0 2023-07-26T09:43:43Z TPR protein PR: cleaner0 2023-07-26T09:42:30Z Cdc23 0.9910953 structure_element cleaner0 2023-07-26T09:51:00Z SO: superhelical structure 0.99880505 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.999269 structure_element cleaner0 2023-07-26T09:13:49Z SO: TPR motif 1 0.99945503 structure_element cleaner0 2023-07-26T09:13:53Z SO: TPR1 protein PR: cleaner0 2023-07-26T09:42:46Z Cdc23 site SO: cleaner0 2023-07-26T12:04:24Z inner groove 0.9081493 structure_element cleaner0 2023-07-26T09:43:42Z SO: TPR 0.9844491 structure_element cleaner0 2023-07-26T09:44:22Z SO: α-helices 0.9981635 evidence cleaner0 2023-07-26T09:44:26Z DUMMY: structure structure_element SO: cleaner0 2023-07-26T09:43:43Z TPR RESULTS paragraph 23397 Self-association of HcpC has not been reported, and there is no domain-swapped structure in the SLR motifs of HcpC, in contrast to that observed in SEL1Lcent. Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A). The interrupted SLR motifs may be required for dimerization of SEL1Lcent, as five SLR motifs are more than enough to form the semicircle of the yin-yang symbol (Fig. 1B). Helix 5A from SLR motif 5 meets helix 9A from SLR motif 9 of the counterpart SEL1L. If the SLR motifs 5 to 9 were not isolated from other SLR motifs, steric hindrance could interfere with dimerization of SEL1L. This is one of the biggest differences from TPRs in Cdc23 and from the SLRs in HcpC, where the motifs exist in tandem. TPR and SLR motifs are generally involved in protein-protein interaction modules, and the sequences between the SLR motifs of SEL1L might actually facilitate the self-association of this protein. 0.99859196 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.9856379 protein_state cleaner0 2023-07-26T09:03:04Z DUMMY: domain-swapped structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9986305 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.9993549 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent 0.9992762 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9985732 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9992106 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR oligomeric_state DUMMY: cleaner0 2023-07-26T11:57:09Z dimerization 0.9993672 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR structure_element SO: cleaner0 2023-07-26T09:15:01Z semicircle of the yin-yang 0.9988102 structure_element cleaner0 2023-07-26T09:49:09Z SO: Helix 5A 0.9962883 structure_element cleaner0 2023-07-26T09:15:11Z SO: SLR motif 5 0.99878836 structure_element cleaner0 2023-07-26T08:40:20Z SO: helix 9A 0.9975462 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.9993511 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.98970985 structure_element cleaner0 2023-07-26T08:25:36Z SO: SLR motifs 5 to 9 structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR oligomeric_state DUMMY: cleaner0 2023-07-26T11:57:09Z dimerization 0.9993968 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.9991345 structure_element cleaner0 2023-07-26T08:40:43Z SO: TPRs 0.9992237 protein cleaner0 2023-07-26T09:10:14Z PR: Cdc23 0.9994486 structure_element cleaner0 2023-07-26T08:41:13Z SO: SLRs 0.9989011 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.9992543 structure_element cleaner0 2023-07-26T09:43:43Z SO: TPR structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.99938834 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L RESULTS title_2 24423 SLR-C of SEL1L Binds HRD1 N-terminus Luminal Loop 0.99893826 structure_element cleaner0 2023-07-26T08:33:22Z SO: SLR-C 0.9991726 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.7173637 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.9890427 structure_element cleaner0 2023-07-26T09:47:49Z SO: Luminal Loop RESULTS paragraph 24473 Based on the structural data presented herein, a possible arrangement of membrane-associated ERAD components in mammals, highlighting the molecular functions of SLR domains in SEL1L, is shown in Fig. 6C. We suggest that the middle SLR domains are involved in the dimerization of SEL1L based on the crystal structure and biochemical data. SLR-C, which contains SLR motifs 10 to 11, might be involved in the interaction with HRD1. Indirect evidence from a previous yeast study shows that the circumscribed region of C-terminal Hrd3p, specifically residues 664–695, forms contacts with the Hrd1 luminal loops. The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). To address this hypothesis, we prepared constructs encoding mouse HRD1 luminal fragments fused to GST as shown in Fig. 6A, and tested their ability to bind certain SLR motifs in SEL1L. The fusion proteins were immobilized on glutathione-Sepharose beads and probed for binding to SLR-N, SLR-M, SLR-C, and monomer form of SLR-M (SLR-ML521A). Figure 6B shows that the SLR-C, consisting of SLR motifs 10 and 11, exclusively interacts with N-terminal luminal loop (residues 21–42) of HRD1. 0.99301314 evidence cleaner0 2023-07-26T11:55:02Z DUMMY: structural data 0.99872905 taxonomy_domain cleaner0 2023-07-26T11:55:33Z DUMMY: mammals 0.99515957 structure_element cleaner0 2023-07-26T08:31:37Z SO: SLR 0.99928576 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR oligomeric_state DUMMY: cleaner0 2023-07-26T11:57:09Z dimerization 0.99938464 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.99822503 evidence cleaner0 2023-07-26T11:52:55Z DUMMY: crystal structure structure_element SO: cleaner0 2023-07-26T08:33:22Z SLR-C 0.9579142 structure_element cleaner0 2023-07-26T09:51:05Z SO: SLR motifs 10 to 11 0.50802 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.9944859 taxonomy_domain cleaner0 2023-07-26T08:27:26Z DUMMY: yeast 0.9987024 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p 0.9977992 residue_range cleaner0 2023-07-26T09:16:48Z DUMMY: 664–695 0.92809564 protein cleaner0 2023-07-26T08:26:25Z PR: Hrd1 0.99864084 structure_element cleaner0 2023-07-26T09:51:10Z SO: luminal loops 0.99743974 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p 0.99779993 residue_range cleaner0 2023-07-26T09:16:51Z DUMMY: 664–695 0.9900275 taxonomy_domain cleaner0 2023-07-26T08:29:39Z DUMMY: mouse 0.9992859 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.9977267 residue_range cleaner0 2023-07-26T09:16:53Z DUMMY: 696–727 0.9986229 structure_element cleaner0 2023-07-26T09:17:17Z SO: helix 11B 0.99773 residue_range cleaner0 2023-07-26T09:16:56Z DUMMY: 697–709 0.99542075 structure_element cleaner0 2023-07-26T09:17:14Z SO: SLR motif 11 0.99896866 protein_state cleaner0 2023-07-26T09:16:58Z DUMMY: well-conserved 0.9705908 structure_element cleaner0 2023-07-26T09:17:21Z SO: SLR motif 10 to 11 0.9988179 experimental_method cleaner0 2023-07-26T12:20:42Z MESH: structure-guided SLR motif alignment 0.82161427 experimental_method cleaner0 2023-07-26T12:20:46Z MESH: structure study 0.7311754 protein_state cleaner0 2023-07-26T12:01:37Z DUMMY: sequence conservation 0.99881953 taxonomy_domain cleaner0 2023-07-26T08:25:52Z DUMMY: mammalian 0.99931455 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.9988205 taxonomy_domain cleaner0 2023-07-26T08:27:26Z DUMMY: yeast 0.95084345 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p 0.98013896 structure_element cleaner0 2023-07-26T09:17:31Z SO: SLR motifs 10 to 11 0.73068887 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.9921606 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.99814737 taxonomy_domain cleaner0 2023-07-26T08:29:39Z DUMMY: mouse 0.9586618 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 experimental_method MESH: cleaner0 2023-07-26T09:17:58Z fused to GST structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.99931836 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.95016915 structure_element cleaner0 2023-07-26T08:35:33Z SO: SLR-N 0.95825976 structure_element cleaner0 2023-07-26T08:35:41Z SO: SLR-M 0.98809576 structure_element cleaner0 2023-07-26T08:33:22Z SO: SLR-C 0.99815935 oligomeric_state cleaner0 2023-07-26T08:39:02Z DUMMY: monomer 0.98904943 structure_element cleaner0 2023-07-26T08:35:41Z SO: SLR-M 0.9985962 mutant cleaner0 2023-07-26T09:18:25Z MESH: SLR-ML521A structure_element SO: cleaner0 2023-07-26T08:33:22Z SLR-C 0.9908142 structure_element cleaner0 2023-07-26T08:33:26Z SO: SLR motifs 10 and 11 0.99880993 structure_element cleaner0 2023-07-26T09:47:49Z SO: luminal loop 0.9976925 residue_range cleaner0 2023-07-26T09:18:30Z DUMMY: 21–42 0.591227 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 RESULTS paragraph 26172 The molecular functions of SLR-N are unclear. One possibility is that SLR-N contributes to substrate recognition of proteins to be degraded because there are a couple of putative glycosylation sites within the SLR-N domain (Fig. 1A). SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C). 0.9861813 structure_element cleaner0 2023-07-26T08:35:33Z SO: SLR-N 0.7683429 structure_element cleaner0 2023-07-26T08:35:33Z SO: SLR-N site SO: cleaner0 2023-07-26T09:19:34Z glycosylation sites structure_element SO: cleaner0 2023-07-26T08:35:33Z SLR-N 0.99923563 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent site SO: cleaner0 2023-07-26T09:19:45Z N-glycosylation site 0.9950211 residue_name_number cleaner0 2023-07-26T09:19:48Z DUMMY: Asn 427 0.9987097 protein_state cleaner0 2023-07-26T09:19:51Z DUMMY: highly conserved 0.9993482 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.9988103 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.99884605 evidence cleaner0 2023-07-26T11:52:55Z DUMMY: crystal structure DISCUSS title_1 26622 Discussion DISCUSS paragraph 26633 Many reports demonstrate that membrane-bound ERAD machinery proteins in yeast, such as Hrd1p, Der1p, and Usa1p, are involved in oligomerization of ERAD components. The Hrd1p complex forms dimers upon sucrose gradient sedimentation and size-exclusion chromatography. Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. Considering that the functional and structural composition of ERAD components are conserved in both yeast and mammals, we propose that the mammalian ERAD components also form self-associating oligomers. This hypothesis is supported by cross-linking data suggesting that human HRD1 forms a homodimer. Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1Lcent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9. We need to further test whether there are contacts involved in dimer formation in SEL1L in addition to those in the SLR-M region. 0.9989015 taxonomy_domain cleaner0 2023-07-26T08:27:26Z DUMMY: yeast 0.9981091 protein cleaner0 2023-07-26T09:20:04Z PR: Hrd1p 0.99841964 protein cleaner0 2023-07-26T08:26:42Z PR: Der1p 0.99871147 protein cleaner0 2023-07-26T08:27:00Z PR: Usa1p 0.99583495 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.9987563 oligomeric_state cleaner0 2023-07-26T11:56:32Z DUMMY: dimers 0.9988889 experimental_method cleaner0 2023-07-26T12:21:08Z MESH: sucrose gradient sedimentation 0.99879324 experimental_method cleaner0 2023-07-26T12:21:11Z MESH: size-exclusion chromatography 0.9873705 protein_state cleaner0 2023-07-26T09:20:21Z DUMMY: HA-epitope-tagged 0.9904461 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p 0.97846574 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.99916005 protein_state cleaner0 2023-07-26T09:20:24Z DUMMY: unmodified 0.99035174 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p 0.9786226 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.98326415 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.986401 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p 0.99876297 oligomeric_state cleaner0 2023-07-26T08:39:18Z DUMMY: homodimers 0.97608227 complex_assembly cleaner0 2023-07-26T09:24:15Z GO: Hrd 0.9987778 taxonomy_domain cleaner0 2023-07-26T08:27:26Z DUMMY: yeast 0.99861693 taxonomy_domain cleaner0 2023-07-26T11:55:38Z DUMMY: mammals 0.9984384 taxonomy_domain cleaner0 2023-07-26T08:25:52Z DUMMY: mammalian 0.99883693 oligomeric_state cleaner0 2023-07-26T11:56:36Z DUMMY: oligomers 0.7436882 experimental_method cleaner0 2023-07-26T12:21:15Z MESH: cross-linking data 0.99834514 species cleaner0 2023-07-26T12:25:26Z MESH: human 0.997649 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.9987276 oligomeric_state cleaner0 2023-07-26T08:24:28Z DUMMY: homodimer 0.9978006 evidence cleaner0 2023-07-26T11:52:55Z DUMMY: crystal structure 0.92263424 evidence cleaner0 2023-07-26T11:55:09Z DUMMY: biochemical data 0.995531 taxonomy_domain cleaner0 2023-07-26T08:29:39Z DUMMY: mouse 0.99918634 structure_element cleaner0 2023-07-26T08:23:47Z SO: SEL1Lcent 0.9987508 oligomeric_state cleaner0 2023-07-26T08:24:28Z DUMMY: homodimer 0.99854213 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.9988674 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.99924845 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:35:41Z SLR-M DISCUSS paragraph 27746 In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity. However, metazoans lack a clear Usa1p homolog. Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p. Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1. This is further supported by our data showing that the SLR-C of SEL1L directly interacts with the luminal fragment of HRD1 in the ER lumen. 0.99887055 taxonomy_domain cleaner0 2023-07-26T08:27:26Z DUMMY: yeast 0.99914896 protein cleaner0 2023-07-26T08:27:00Z PR: Usa1p 0.99887496 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.99909854 protein cleaner0 2023-07-26T08:26:42Z PR: Der1p 0.9991042 protein cleaner0 2023-07-26T08:27:00Z PR: Usa1p 0.9988599 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.9987087 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.99869114 taxonomy_domain cleaner0 2023-07-26T08:27:33Z DUMMY: metazoans 0.9989818 protein cleaner0 2023-07-26T08:27:00Z PR: Usa1p 0.9985386 taxonomy_domain cleaner0 2023-07-26T08:25:52Z DUMMY: mammalian 0.9656231 protein_type cleaner0 2023-07-26T09:46:58Z MESH: HERP 0.8323746 protein_state cleaner0 2023-07-26T12:01:47Z DUMMY: conserved in 0.99918956 protein cleaner0 2023-07-26T08:27:00Z PR: Usa1p 0.98418415 protein_type cleaner0 2023-07-26T09:46:59Z MESH: HERP 0.99921775 protein cleaner0 2023-07-26T08:27:00Z PR: Usa1p 0.6903281 protein_state cleaner0 2023-07-26T09:21:44Z DUMMY: transiently expressed 0.9989593 complex_assembly cleaner0 2023-07-26T08:32:43Z GO: HRD1-SEL1L 0.8359795 protein_type cleaner0 2023-07-26T12:25:19Z MESH: lectins 0.9986066 protein cleaner0 2023-07-26T08:27:05Z PR: OS9 0.997894 protein cleaner0 2023-07-26T08:27:14Z PR: XTP-B protein PR: cleaner0 2023-07-26T09:22:22Z α-antitrypsin null Hong-Kong 0.98373795 protein cleaner0 2023-07-26T09:22:35Z PR: NHK 0.998617 mutant cleaner0 2023-07-26T12:27:17Z MESH: NHK-QQQ 0.99814117 protein_state cleaner0 2023-07-26T12:01:57Z DUMMY: lacks 0.99818313 site cleaner0 2023-07-26T09:21:19Z SO: N-glycosylation sites 0.9988011 oligomeric_state cleaner0 2023-07-26T08:24:28Z DUMMY: homodimer 0.99926955 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.99525696 complex_assembly cleaner0 2023-07-26T09:24:15Z GO: HRD 0.99923074 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L protein_state DUMMY: cleaner0 2023-07-26T12:27:01Z complex with 0.9989691 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.99814767 structure_element cleaner0 2023-07-26T08:33:22Z SO: SLR-C 0.9991585 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.9979825 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 DISCUSS paragraph 28919 Although the organization of membrane-bound HRD complex components may be very similar between metazoans and yeast, the molecular details of interactions between the components may not necessarily be conserved. In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L. Furthermore, we are uncertain whether self-association of Hrd3p contributes to formation of the active form of the Hrd1p complex. Recently, a truncated version of Yos9 was shown to form a dimer in the ER lumen and to contribute to the dimeric state of the Hrd1p complex. This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9. However, the dimerization of Yos9 could provide a higher stability for the Hrd1p complex oligomer. Likewise, the dimerization of SEL1L might provide stability for the mammalian HRD oligomer complex. Further cell biological studies are required to clarify whether SEL1L (Hrd3p) dimerization could be cooperative with the oligomerization of the HRD complex. 0.7623892 complex_assembly cleaner0 2023-07-26T09:24:15Z GO: HRD 0.9986921 taxonomy_domain cleaner0 2023-07-26T08:27:33Z DUMMY: metazoans 0.998847 taxonomy_domain cleaner0 2023-07-26T08:27:26Z DUMMY: yeast 0.9988938 taxonomy_domain cleaner0 2023-07-26T08:27:26Z DUMMY: yeast 0.9938378 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9938076 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.99895895 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.98880565 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p 0.9991412 protein_state cleaner0 2023-07-26T12:02:08Z DUMMY: active 0.79716533 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.9986615 protein_state cleaner0 2023-07-26T12:02:11Z DUMMY: truncated 0.9987722 protein cleaner0 2023-07-26T08:27:09Z PR: Yos9 0.99889237 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9989335 oligomeric_state cleaner0 2023-07-26T11:56:43Z DUMMY: dimeric 0.72566545 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.91094744 protein cleaner0 2023-07-26T08:27:09Z PR: Yos9 0.65282786 protein cleaner0 2023-07-26T08:27:09Z PR: Yos9 0.98758864 experimental_method cleaner0 2023-07-26T12:21:23Z MESH: immunoprecipitation experiments 0.9987257 taxonomy_domain cleaner0 2023-07-26T08:27:26Z DUMMY: yeast protein_state DUMMY: cleaner0 2023-07-26T09:23:32Z epitope-tagged 0.9984559 protein cleaner0 2023-07-26T08:27:09Z PR: Yos9 oligomeric_state DUMMY: cleaner0 2023-07-26T11:57:09Z dimerization 0.99885285 protein cleaner0 2023-07-26T08:27:09Z PR: Yos9 0.68382525 protein cleaner0 2023-07-26T09:20:05Z PR: Hrd1p 0.99890447 oligomeric_state cleaner0 2023-07-26T11:57:18Z DUMMY: oligomer 0.99372673 oligomeric_state cleaner0 2023-07-26T11:57:08Z DUMMY: dimerization 0.9992169 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.9986565 taxonomy_domain cleaner0 2023-07-26T08:25:52Z DUMMY: mammalian 0.9694277 complex_assembly cleaner0 2023-07-26T09:24:15Z GO: HRD 0.99869686 oligomeric_state cleaner0 2023-07-26T11:57:18Z DUMMY: oligomer 0.9991221 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.9679804 protein cleaner0 2023-07-26T08:26:30Z PR: Hrd3p 0.93076116 oligomeric_state cleaner0 2023-07-26T11:57:09Z DUMMY: dimerization 0.5775222 complex_assembly cleaner0 2023-07-26T09:24:15Z GO: HRD DISCUSS paragraph 30125 Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. These findings should provide a foundation for molecular-level studies to understand the membrane-associated HRD complex assembly in ERAD. 0.6018751 complex_assembly cleaner0 2023-07-26T09:24:14Z GO: HRD 0.9982066 oligomeric_state cleaner0 2023-07-26T11:57:23Z DUMMY: oligomers 0.9993987 protein cleaner0 2023-07-26T08:24:01Z PR: SEL1L 0.998985 protein_state cleaner0 2023-07-26T12:02:15Z DUMMY: active 0.58802515 complex_assembly cleaner0 2023-07-26T09:24:15Z GO: HRD 0.9988446 protein_state cleaner0 2023-07-26T12:02:21Z DUMMY: absence of 0.99927646 protein cleaner0 2023-07-26T08:27:00Z PR: Usa1p 0.9982326 taxonomy_domain cleaner0 2023-07-26T08:27:33Z DUMMY: metazoans 0.62106395 complex_assembly cleaner0 2023-07-26T09:24:15Z GO: HRD METHODS title_1 30539 Methods METHODS title_2 30547 Protein Production METHODS paragraph 30566 The expression and purification of SEL1L was performed as described previously. METHODS title_2 30646 Crystallization and SAD Structure Determination of SEL1Lcent METHODS paragraph 30707 Crystals were grown using the hanging-drop vapor diffusion method at 4 °C. For crystallization of the M. musculus SEL1Lcent, 1 μl of protein solution (in 25 mM Tris-HCl, 150 mM NaCl, and 5 mM DTT, pH 7.5) was equilibrated with 1 μl of well solution (30% isopropanol, 100 mM NaCl, 100 mM Tris, 5 mM DTT, and 20 mM phenol, pH 8.5). The crystals, which appeared after 4 days, contain two SEL1Lcent dimers in the asymmetric unit (space group P21, a = 29.13, b = 110.52, c = 109.81 Å, α = 90.00, β = 90.61, γ = 90.00, 44% solvent). For X-ray diffraction experiments, crystals were transferred to well solution plus paraffin-oil, then flash frozen in liquid nitrogen. METHODS paragraph 31422 SAD data were collected with a Se-Met crystal at beamline 7A of the Pohang Accelerator Laboratory (PAL) and processed using HKL2000 software. Native data (2.6 Å resolution) were collected from a single frozen crystal at the same beamline of PAL and were integrated and scaled as described above. The SAD data analysis was performed using Phenix software using data between 50 and 2.9 Å resolution. Phenix identified 31 of the 32 selenium sites and refined these to give a mean f.o.m. = 0.472. Electron density modification, including non-crystallographic symmetry (NCS) averaging, using the RESOLVE software yielded an initial electron density map of excellent quality. Model building and refinement were carried out with the Coot and Phenix programs, respectively. The final model was refined to an R factor of 20.7% (Rfree = 27.7%) for native data between 30 and 2.6 Å resolution (Table 1). The final model consisted of 5402 protein atoms and 47 water molecules. There were no outliers in a Ramachandran plot of the final model. The model contained four copies of SEL1Lcent (residues 348–533) in the asymmetric unit. Of these, the following residues were not modeled due to weak electron densities: SEL1Lcent residues 348–351, 420, 421, and 525–533 in the first copy; residues 348–351 and 525–533 in the second and third copies; and residues 348–352 and 525–533 in the fourth copy. The X-ray data and refinement statistics are summarized in Table 1. METHODS title_2 32905 Cell Culture and Plasmids Construction METHODS paragraph 32944 HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS. The mouse Sel1L gene was cloned into pCS108 and the 3 × HA or 3 × FLAG tag was fused to the C-terminus of SEL1L. The signal peptide from Xenopus Sel1L was cloned into pCS108 and the mouse SEL1Lcent domain, SEL1L (348-497) fragments, and SEL1Lcent (L521A) were fused to the C-terminus of the signal peptide. Then, a 3 × HA or a 3 × FLAG tag was fused to the C-terminus of the constructs. For the ER retention signal, the KDEL sequences were added to the C-terminus of the fragments. The plasmids were transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s manual. METHODS title_2 33634 Western Blot Analysis and Immunostaining METHODS paragraph 33675 For western blot analysis, HEK293T cells were transfected with the indicated construct and harvested after washing in PBS. The cells were homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 5% glycerol), supplemented with protease and phosphatase inhibitor cocktails. Homogenates were cleared by centrifugation at 13,200 rpm for 15 minutes at 4 °C. The lysates were subsequently used for either co-immunoprecipitation experiment or western blot analysis. For the western blot analysis, the samples were run onto 6–12% polyacrylamide gel. Blots were blocked in 5% TBS + 0.05% Tween 20 and incubated with anti-DDDD-K (Abcam) or anti-HA (Roche) antibodies. Proteins were visualized using HRP-conjugated secondary antibodies (1:4000) and SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo) and exposed to ChemiDoc MP (Bio-Rad). METHODS paragraph 34604 For immunostaining, the cells were fixed in 4% formaldehyde and incubated with the indicated antibodies. The coverslips were incubated in blocking solution (10% FBS + 2% DMSO in TBS + 0.1% Triton X-100) at room temperature for 30 minutes to block non-specific binding. Fluorescent labeling was performed using Alexa Fluor 555 or 488-conjugated secondary antibodies and nuclei were stained with DAPI. The samples were mounted and confocal images were obtained using a Zeiss LSM700. METHODS title_2 35095 GST Pull-down Assay METHODS paragraph 35115 For pull-down experiments, 400 μg of HRD1 luminal fragment GST-fusion proteins were incubated with 5 μl of a 50% (v/v) slurry of glutathione sepharose 4B beads (GE Healthcare) for 50 min at 4 °C. Beads were washed twice with buffer A (150 mM NaCl, 25 mM sodium phosphate pH 7.5, 5 mM DTT), and then mixed with 100 μg of MBP-SEL1L protein (SLR-N, SLR-M, SLR-C, and SLR-ML521A) in buffer A, in a total assay volume of 500 μl. The assay mix was incubated at 4 °C for 15 minutes, and beads were washed twice with 500 μl buffer A. Proteins were eluted with SDS sample buffer, and analyzed by SDS-PAGE. METHODS title_1 35743 Additional Information METHODS paragraph 35766 Accession Numbers: The coordinates and structure factors have been deposited in the Protein Data Bank with the accession code of 5B26. METHODS paragraph 35901 How to cite this article: Jeong, H. et al. Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway. Sci. Rep. 6, 20261; doi: 10.1038/srep20261 (2016). SUPPL title_1 36077 Supplementary Material 333 366 surname:Chiti;given-names:F. surname:Dobson;given-names:C. 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A. 21074049 REF Cell ref 143 2010 39013 Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p 16929 16939 surname:Iida;given-names:Y. 21454652 REF J Biol Chem ref 286 2011 39096 SEL1L protein critically determines the stability of the HRD1-SEL1L endoplasmic reticulum-associated degradation (ERAD) complex to optimize the degradation kinetics of ERAD substrates 8633 8640 surname:Hanna;given-names:J. 22262864 REF J Biol Chem ref 287 2012 39280 Structural and biochemical basis of Yos9 protein dimerization and possible contribution to self-association of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation ubiquitin-ligase complex 1624 1627 surname:Jeong;given-names:H. surname:Lee;given-names:H. surname:Lee;given-names:C. REF Acta Crystallogr F ref 70 2014 39476 Crystallization and preliminary X-ray diffraction analysis of the Sel1-like repeats of SEL1L 307 326 surname:Otwinowski;given-names:Z. surname:Minor;given-names:W. REF Method Enzymol ref 276 1997 39569 Processing of X-ray diffraction data collected in oscillation mode 213 221 surname:Adams;given-names:P. D. 20124702 REF Acta Crystallogr D ref 66 2010 39636 PHENIX: a comprehensive Python-based system for macromolecular structure solution 849 861 surname:Terwilliger;given-names:T. C. surname:Berendzen;given-names:J. 10089316 REF Acta Crystallogr D ref 55 1999 39718 Automated MAD and MIR structure solution 486 501 surname:Emsley;given-names:P. surname:Lohkamp;given-names:B. surname:Scott;given-names:W. G. surname:Cowtan;given-names:K. 20383002 REF Acta Crystallogr D ref 66 2010 39759 Features and development of Coot SUPPL footnote 39792 Author Contributions H.J. and H.L. performed cloning, expression, and protein purification. H.J., S.H. and C.L. crystallized, collected X-ray data, and solved the protein structure. H.J., H.S., E.S. and Y.J performed biochemical experiments. H.J., T.P. and C.L. wrote the manuscript. T.P. and C.L. designed and supervised the project. All the authors discussed the results, commented on the manuscript, and approved the manuscript. srep20261-f1.jpg f1 FIG fig_title_caption 40224 Crystal Structure of SEL1Lcent. 0.9985441 evidence cleaner0 2023-07-26T11:52:56Z DUMMY: Crystal Structure 0.9989202 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent srep20261-f1.jpg f1 FIG fig_caption 40256 (A) The diagram shows the domain structure of Mus musculus SEL1L, as defined by proteolytic mapping and sequence/structure analysis. The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs. Putative N-glycosylation sites are indicated by black triangles. We determined the crystal structure of the SLR-M, residues 348–533. (B) Ribbon diagram of the biological unit of the SEL1Lcent, viewed along the two-fold NCS axis. The crystal structure was determined by SAD phasing using selenium as the anomalous scatterer and refined to 2.6 Å resolution (Table 1). (C) SEL1Lcent ribbon diagram rotated 90° around a horizontal axis relative to (B). (D) One protomer of the SEL1Lcent dimer. This view is rotated about 90° anticlockwise from the bottom copy in (B), along the two-fold NCS axis. Starting from the N-terminus, SEL1Lcent has five SLR motifs comprising ten α helices. Each SLR motif (from 5 to 9) is indicated in a different color. (E) Evolutionary conservation of surface residues in SEL1Lcent, calculated using ConSurf, from a structure-based alignment of 135 SEL1L sequences. The surface is colored from red (high) to white (poor) according to the degree of conservation in the SEL1L phylogenetic orthologs. The ribbon diagram of the counterpart protomer is drawn to show the orientation of the SEL1Lcent dimer. 0.99830663 species cleaner0 2023-07-26T08:34:15Z MESH: Mus musculus 0.99923646 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.9986738 experimental_method cleaner0 2023-07-26T12:21:30Z MESH: proteolytic mapping 0.9985619 experimental_method cleaner0 2023-07-26T12:21:35Z MESH: sequence/structure analysis structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.99073833 structure_element cleaner0 2023-07-26T08:35:33Z SO: SLR-N 0.9909288 structure_element cleaner0 2023-07-26T08:35:42Z SO: SLR-M 0.9904032 structure_element cleaner0 2023-07-26T08:33:22Z SO: SLR-C 0.997459 structure_element cleaner0 2023-07-26T08:35:21Z SO: linker sequences structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.99653506 site cleaner0 2023-07-26T09:30:56Z SO: N-glycosylation sites 0.99770147 evidence cleaner0 2023-07-26T11:52:56Z DUMMY: crystal structure 0.8388017 structure_element cleaner0 2023-07-26T08:35:42Z SO: SLR-M 0.99781173 residue_range cleaner0 2023-07-26T09:31:05Z DUMMY: 348–533 0.9988219 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.99648273 evidence cleaner0 2023-07-26T11:52:56Z DUMMY: crystal structure 0.99892163 experimental_method cleaner0 2023-07-26T12:21:44Z MESH: SAD phasing 0.99900913 chemical cleaner0 2023-07-26T08:38:45Z CHEBI: selenium 0.9987111 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9989466 oligomeric_state cleaner0 2023-07-26T08:39:07Z DUMMY: protomer 0.99757284 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9988978 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9988696 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9988542 structure_element cleaner0 2023-07-26T09:31:20Z SO: α helices structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.99901414 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.998268 experimental_method cleaner0 2023-07-26T12:21:50Z MESH: ConSurf 0.9988711 experimental_method cleaner0 2023-07-26T12:21:54Z MESH: structure-based alignment 0.9931625 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.99388146 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.99892056 oligomeric_state cleaner0 2023-07-26T08:39:07Z DUMMY: protomer 0.99843997 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.998904 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer srep20261-f2.jpg f2 FIG fig_title_caption 41671 Dimer Interface of SEL1Lcent. 0.99903095 site cleaner0 2023-07-26T08:43:54Z SO: Dimer Interface 0.9991755 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent srep20261-f2.jpg f2 FIG fig_caption 41701 (A) The diagram on the left shows the SEL1Lcent dimer viewed along the two-fold symmetry axis. Three distinct contact regions are indicated with labeled boxes. The close-up view on the right shows the residues of SEL1Lcent that contribute to dimer formation via the three contact interfaces. Oxygen and nitrogen atoms are shown as red and blue, respectively. The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. The standard molecular masses for the SEC experiments (top) were obtained from the following proteins: aldolase, 158 kDa; cobalbumin, 75 kDa; ovalbumin, 44 kDa; and carbonic anhydrase, 29 kDa. Chromatography was performed on a Superdex 200 column with a buffer containing 25 mM Tris, 150 mM NaCl, and 5 mM DTT (pH 7.5). The elution fractions, indicated by the gray shading, were run on SDS-PAGE and are shown below the gel-filtration elution profile. The schematic diagrams representing the protein constructs used in the SEC are shown on the left of each SDS-PAGE profile. 0.99904746 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9988695 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9326523 site cleaner0 2023-07-26T12:04:53Z SO: contact regions 0.99910814 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9988939 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9985516 site cleaner0 2023-07-26T12:04:58Z SO: contact interfaces 0.99579656 bond_interaction cleaner0 2023-07-26T09:31:54Z MESH: hydrogen bonds 0.9982621 oligomeric_state cleaner0 2023-07-26T08:39:13Z DUMMY: protomers 0.9991347 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9989516 experimental_method cleaner0 2023-07-26T12:22:05Z MESH: Size-exclusion chromatography 0.9987413 experimental_method cleaner0 2023-07-26T12:22:10Z MESH: SEC 0.9990842 protein_state cleaner0 2023-07-26T08:53:21Z DUMMY: wild-type site SO: cleaner0 2023-07-26T09:32:46Z dimeric interface 0.9965245 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9806871 protein_state cleaner0 2023-07-26T12:02:31Z DUMMY: mutants 0.99843615 experimental_method cleaner0 2023-07-26T12:22:16Z MESH: SEC 0.99640155 experimental_method cleaner0 2023-07-26T12:22:19Z MESH: SDS-PAGE evidence DUMMY: cleaner0 2023-07-26T12:22:35Z gel-filtration elution profile 0.9978103 experimental_method cleaner0 2023-07-26T12:22:58Z MESH: SEC 0.9939421 experimental_method cleaner0 2023-07-26T12:23:01Z MESH: SDS-PAGE srep20261-f3.jpg f3 FIG fig_title_caption 42903 Domain Swapping for Dimerization of SEL1Lcent. 0.99684453 oligomeric_state cleaner0 2023-07-26T11:57:09Z DUMMY: Dimerization 0.9990489 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent srep20261-f3.jpg f3 FIG fig_caption 42950 (A) Sequence alignment of the SLR motifs in SEL1L. The 11 SLR motifs were aligned based on the present crystal structure of SEL1Lcent. The sequences of SEL1Lcent included in the crystal structure are highlighted by the blue box. The secondary structure elements are indicated above the sequences, with helices depicted as cylinders. Residues that are conserved in at least 7 out of 11 sequences are red. The GG sequence in SLR motif 9, which creates the hinge for domain swapping (see text), is shaded yellow. Stars below the sequences indicate the specific residues that commonly appear in SLRs. (B) Structure alignment of five SLR motifs in SEL1Lcent is shown to highlight the unusual geometry of SLR motif 9. Each SLR motif is shown in a different color. The arrow indicates the direction of the helical axes. In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. Oxygen and nitrogen atoms are colored red and blue, respectively. The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. Size-exclusion chromatography was conducted as described in Fig. 2B. The standard molecular masses are shown at the top as in Fig. 2B. 0.9987904 experimental_method cleaner0 2023-07-26T12:23:20Z MESH: Sequence alignment structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9992772 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9936063 experimental_method cleaner0 2023-07-26T12:23:26Z MESH: aligned 0.99686766 evidence cleaner0 2023-07-26T11:52:56Z DUMMY: crystal structure 0.9993211 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9993175 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9664356 evidence cleaner0 2023-07-26T11:52:56Z DUMMY: crystal structure 0.70380676 structure_element cleaner0 2023-07-26T09:51:23Z SO: helices 0.5623606 structure_element cleaner0 2023-07-26T09:33:39Z SO: GG 0.9946766 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.99734586 structure_element cleaner0 2023-07-26T09:00:43Z SO: hinge 0.99871385 structure_element cleaner0 2023-07-26T08:41:13Z SO: SLRs 0.9989205 experimental_method cleaner0 2023-07-26T12:23:31Z MESH: Structure alignment structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9992717 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9945833 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.99119973 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.9149917 structure_element cleaner0 2023-07-26T09:51:26Z SO: helices structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9991172 structure_element cleaner0 2023-07-26T09:51:29Z SO: α-hairpin 0.9924176 residue_name_number cleaner0 2023-07-26T09:34:14Z DUMMY: Gly 512 0.995074 residue_name_number cleaner0 2023-07-26T09:34:18Z DUMMY: Gly 513 structure_element SO: cleaner0 2023-07-26T08:41:50Z helix 9B 0.9989567 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer 0.9913488 residue_name_number cleaner0 2023-07-26T09:34:15Z DUMMY: Gly 512 0.9942075 residue_name_number cleaner0 2023-07-26T09:34:19Z DUMMY: Gly 513 0.99725544 experimental_method cleaner0 2023-07-26T12:23:38Z MESH: point mutations 0.9937947 residue_name_number cleaner0 2023-07-26T09:34:15Z DUMMY: Gly 512 0.99478316 residue_name_number cleaner0 2023-07-26T09:34:19Z DUMMY: Gly 513 0.9989575 structure_element cleaner0 2023-07-26T09:00:43Z SO: hinge 0.99069184 structure_element cleaner0 2023-07-26T08:25:42Z SO: SLR motif 9 0.9985311 mutant cleaner0 2023-07-26T09:02:13Z MESH: G512A 0.9986308 mutant cleaner0 2023-07-26T09:02:18Z MESH: G513A 0.9983796 mutant cleaner0 2023-07-26T09:02:13Z MESH: G512A 0.99881685 mutant cleaner0 2023-07-26T09:02:18Z MESH: G513A 0.9988588 mutant cleaner0 2023-07-26T09:02:43Z MESH: G512K 0.99901724 mutant cleaner0 2023-07-26T09:02:48Z MESH: G513K 0.9989951 experimental_method cleaner0 2023-07-26T12:23:43Z MESH: Size-exclusion chromatography srep20261-f4.jpg f4 FIG fig_title_caption 44504 SEL1L forms self-oligomer mediated by the SEL1Lcent domain in vivo. 0.9991986 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.52479684 oligomeric_state cleaner0 2023-07-26T11:59:16Z DUMMY: self-oligomer 0.99886084 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent srep20261-f4.jpg f4 FIG fig_caption 44572 (A) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. The SEL1L348–497 fragment was secreted to the culture media but the SEL1Lcent was retained in the ER. (C) SEL1Lcent-FLAG-KDEL and SEL1L348–497-FLAG-KDEL localized to the ER. The nuclei were stained with DAPI in blue. The ER was visualized with the anti-calnexin antibody in green. The SEL1L fragments were stained in red. (D) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-HA antibody followed by Western blot analysis using an anti-FLAG antibody. The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. The red asterisk indicates the expected signal for SEL1L348–497-FLAG-KDEL. SEL1L348–497-FLAG-KDEL did not co-immunoprecipitate with full-length SEL1L-HA. The white asterisks indicate non-specific bands. (E) SEL1Lcent-HA-KDEL competitively inhibited self-oligomerization of full-length SEL1L. The indicated plasmid constructs were transfected and immunoprecipitation assay was performed using an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L. 0.9971973 experimental_method cleaner0 2023-07-26T12:23:51Z MESH: immunoprecipitated experimental_method MESH: cleaner0 2023-07-26T09:03:45Z FLAG 0.9885245 experimental_method cleaner0 2023-07-26T12:23:55Z MESH: western blot experimental_method MESH: cleaner0 2023-07-26T09:09:28Z HA 0.99912053 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.9982527 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L experimental_method MESH: cleaner0 2023-07-26T09:03:45Z FLAG 0.9940212 experimental_method cleaner0 2023-07-26T12:23:59Z MESH: co-immunoprecipitated 0.9991062 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.99856913 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.61573565 experimental_method cleaner0 2023-07-26T09:09:28Z MESH: HA 0.997417 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9880214 experimental_method cleaner0 2023-07-26T12:24:02Z MESH: co-immunoprecipitated 0.99909383 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.9989844 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:25:42Z SLR motif 9 0.42224488 experimental_method cleaner0 2023-07-26T12:24:05Z MESH: deletion 0.99481857 experimental_method cleaner0 2023-07-26T12:24:08Z MESH: western blot 0.9980458 experimental_method cleaner0 2023-07-26T12:24:12Z MESH: immunoprecipitation mutant MESH: cleaner0 2023-07-26T09:04:56Z SEL1L348–497 0.99846137 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent mutant MESH: cleaner0 2023-07-26T09:35:10Z SEL1Lcent-FLAG-KDEL mutant MESH: cleaner0 2023-07-26T09:35:44Z SEL1L348–497-FLAG-KDEL 0.3403473 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.99533045 experimental_method cleaner0 2023-07-26T12:24:17Z MESH: immunoprecipitated experimental_method MESH: cleaner0 2023-07-26T09:09:28Z HA 0.9932866 experimental_method cleaner0 2023-07-26T12:24:20Z MESH: Western blot experimental_method MESH: cleaner0 2023-07-26T09:03:45Z FLAG 0.99895054 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.99914587 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L oligomeric_state DUMMY: cleaner0 2023-07-26T09:06:04Z self-oligomers mutant MESH: cleaner0 2023-07-26T09:36:31Z SEL1Lcent-FLAG-KDEL 0.9982154 experimental_method cleaner0 2023-07-26T12:24:24Z MESH: co-immunoprecipitated 0.9989381 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.99822336 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.70323896 experimental_method cleaner0 2023-07-26T09:09:28Z MESH: HA 0.8285012 mutant cleaner0 2023-07-26T09:35:45Z MESH: SEL1L348–497-FLAG-KDEL mutant MESH: cleaner0 2023-07-26T09:35:45Z SEL1L348–497-FLAG-KDEL 0.9989348 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.99864477 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.5467928 experimental_method cleaner0 2023-07-26T09:09:28Z MESH: HA mutant MESH: cleaner0 2023-07-26T09:36:58Z SEL1Lcent-HA-KDEL 0.9990277 protein_state cleaner0 2023-07-26T08:24:14Z DUMMY: full-length 0.99917597 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.99865973 experimental_method cleaner0 2023-07-26T12:24:28Z MESH: immunoprecipitation assay experimental_method MESH: cleaner0 2023-07-26T09:03:45Z FLAG 0.95454943 experimental_method cleaner0 2023-07-26T12:24:30Z MESH: western blot experimental_method MESH: cleaner0 2023-07-26T09:09:28Z HA 0.9990332 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L oligomeric_state DUMMY: cleaner0 2023-07-26T11:57:18Z oligomer mutant MESH: cleaner0 2023-07-26T09:36:59Z SEL1Lcent-HA-KDEL 0.9991394 mutant cleaner0 2023-07-26T08:52:49Z MESH: L521A 0.9413898 protein_state cleaner0 2023-07-26T12:02:37Z DUMMY: point mutant 0.9989951 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent 0.9991678 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L srep20261-f5.jpg f5 FIG fig_title_caption 46507 Comparison of SLR in SEL1L with TPR or Other SLR-Containing Proteins. 0.9993629 structure_element cleaner0 2023-07-26T08:31:37Z SO: SLR 0.9993555 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.9819749 structure_element cleaner0 2023-07-26T09:43:43Z SO: TPR protein_type MESH: cleaner0 2023-07-26T09:37:53Z SLR-Containing Proteins srep20261-f5.jpg f5 FIG fig_caption 46577 (A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). The SEL1L, Cdc23, and HcpC are colored magenta, green and cyan, respectively. Black arrows indicate the helical axes. The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. Both SEL1Lcent schematics are identically oriented for comparison. The Cα atoms of the residues in each α-solenoid domain are superimposed with a root-mean-squared deviation of 3.3 Å for Cdc23 and SEL1Lcent (left), and 2.5 Å for HcpC and SEL1Lcent (right). SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping. The view is along the two-fold axis. 0.9983388 experimental_method cleaner0 2023-07-26T12:24:35Z MESH: superimposition structure_element SO: cleaner0 2023-07-26T09:43:43Z TPR 0.99884206 protein cleaner0 2023-07-26T09:10:14Z PR: Cdc23 structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9989537 structure_element cleaner0 2023-07-26T08:23:48Z SO: SEL1Lcent structure_element SO: cleaner0 2023-07-26T08:31:37Z SLR 0.9991535 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.9990169 structure_element cleaner0 2023-07-26T08:23:49Z SO: SEL1Lcent 0.9981001 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.9981516 protein cleaner0 2023-07-26T09:10:14Z PR: Cdc23 0.9990277 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.98901033 ptm cleaner0 2023-07-26T09:12:11Z MESH: disulfide bonds 0.9985221 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.9974023 residue_name cleaner0 2023-07-26T12:27:50Z SO: Cys 0.9873022 ptm cleaner0 2023-07-26T12:27:58Z MESH: disulfide bonding 0.99798596 experimental_method cleaner0 2023-07-26T12:24:38Z MESH: superimposition 0.99736744 protein cleaner0 2023-07-26T09:10:14Z PR: Cdc23 0.9981723 structure_element cleaner0 2023-07-26T08:23:49Z SO: SEL1Lcent 0.9984665 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.99831355 structure_element cleaner0 2023-07-26T08:23:49Z SO: SEL1Lcent 0.9990736 structure_element cleaner0 2023-07-26T09:38:15Z SO: α-solenoid domain 0.99877936 structure_element cleaner0 2023-07-26T08:23:49Z SO: SEL1Lcent 0.99905175 structure_element cleaner0 2023-07-26T09:38:15Z SO: α-solenoid domain 0.9977223 experimental_method cleaner0 2023-07-26T12:24:43Z MESH: superimposed 0.9984053 evidence cleaner0 2023-07-26T09:38:21Z DUMMY: root-mean-squared deviation 0.9972836 protein cleaner0 2023-07-26T09:10:14Z PR: Cdc23 0.99782985 structure_element cleaner0 2023-07-26T08:23:49Z SO: SEL1Lcent 0.9985839 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.9982064 structure_element cleaner0 2023-07-26T08:23:49Z SO: SEL1Lcent 0.99795073 structure_element cleaner0 2023-07-26T08:23:49Z SO: SEL1Lcent 0.99767953 protein cleaner0 2023-07-26T09:10:14Z PR: Cdc23 0.99890506 protein cleaner0 2023-07-26T09:10:23Z PR: HcpC 0.7976578 evidence cleaner0 2023-07-26T11:55:17Z DUMMY: structure protein PR: cleaner0 2023-07-26T12:27:35Z Cdc23 0.99864715 structure_element cleaner0 2023-07-26T08:23:49Z SO: SEL1Lcent 0.9988312 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer srep20261-f6.jpg f6 FIG fig_title_caption 47690 The Role of SLR-C in ERAD machinery and Model for the Organization of Proteins in Membrane-Associated ERAD Components. 0.9980087 structure_element cleaner0 2023-07-26T08:33:22Z SO: SLR-C srep20261-f6.jpg f6 FIG fig_caption 47809 (A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L. Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. Proteins were analyzed by 12% SDS-PAGE and Coomassie blue staining. (C) Schematic representation of the organization of metazoan ERAD components in the ER membrane. The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen. The surface representation of SEL1Lcent is placed in the same orientation as that shown in the schematic model to show that the putative N-glycosylation site, residue N427 (indicated in yellow), is exposed on the surface of the protein. The yellow arrow indicates self-association among the respective components. 0.9831434 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.99888813 experimental_method cleaner0 2023-07-26T12:24:48Z MESH: GST pull-down 0.98592913 experimental_method cleaner0 2023-07-26T12:24:53Z MESH: Pull-down experiments 0.99947685 complex_assembly cleaner0 2023-07-26T09:24:15Z GO: HRD 0.9987311 structure_element cleaner0 2023-07-26T09:51:11Z SO: luminal loops structure_element SO: cleaner0 2023-07-26T08:31:38Z SLR 0.9991185 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.9990668 structure_element cleaner0 2023-07-26T09:47:49Z SO: luminal loop 0.73801214 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.42447874 chemical cleaner0 2023-07-26T09:39:31Z CHEBI: GST structure_element SO: cleaner0 2023-07-26T08:31:38Z SLR 0.9988919 oligomeric_state cleaner0 2023-07-26T08:39:02Z DUMMY: monomer 0.82218844 structure_element cleaner0 2023-07-26T08:35:42Z SO: SLR-M 0.9988654 mutant cleaner0 2023-07-26T09:18:26Z MESH: SLR-ML521A 0.99908733 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L 0.9981499 experimental_method cleaner0 2023-07-26T12:25:00Z MESH: SDS-PAGE 0.9984635 taxonomy_domain cleaner0 2023-07-26T11:55:43Z DUMMY: metazoan structure_element SO: cleaner0 2023-07-26T08:31:38Z SLR 0.99918073 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:35:34Z SLR-N structure_element SO: cleaner0 2023-07-26T08:35:42Z SLR-M 0.7746102 structure_element cleaner0 2023-07-26T08:33:22Z SO: SLR-C 0.9975531 experimental_method cleaner0 2023-07-26T12:25:04Z MESH: sequence alignment 0.9978694 evidence cleaner0 2023-07-26T11:52:56Z DUMMY: crystal structure structure_element SO: cleaner0 2023-07-26T08:31:38Z SLR 0.9992436 protein cleaner0 2023-07-26T08:24:02Z PR: SEL1L structure_element SO: cleaner0 2023-07-26T08:35:42Z SLR-M 0.9989221 oligomeric_state cleaner0 2023-07-26T08:24:34Z DUMMY: dimer structure_element SO: cleaner0 2023-07-26T08:33:22Z SLR-C 0.9812152 protein cleaner0 2023-07-26T08:26:25Z PR: HRD1 0.9988286 structure_element cleaner0 2023-07-26T08:23:49Z SO: SEL1Lcent 0.9985453 site cleaner0 2023-07-26T12:05:05Z SO: N-glycosylation site 0.99954575 residue_name_number cleaner0 2023-07-26T09:40:08Z DUMMY: N427 t1.xml t1 TABLE table_title_caption 49144 Data Collection and Refinement Statistics. t1.xml t1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/></colgroup><tbody valign="top"><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">SEL1L^cent</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Data set:</td><td align="center" valign="top" charoff="50">Native</td><td align="center" valign="top" charoff="50">Se-SAD</td></tr><tr><td align="left" valign="top" charoff="50">PDB accession #:</td><td align="center" valign="top" charoff="50">5B26</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">X-ray source</td><td align="center" valign="top" charoff="50">Beamline 7A, PAL</td><td align="center" valign="top" charoff="50">Beamline 7A, PAL</td></tr><tr><td align="left" valign="top" charoff="50">Temperature (K)</td><td align="center" valign="top" charoff="50">100</td><td align="center" valign="top" charoff="50">100</td></tr><tr><td align="left" valign="top" charoff="50">Space group:</td><td align="center" valign="top" charoff="50">P2₁</td><td align="center" valign="top" charoff="50">P2₁</td></tr><tr><td align="left" valign="top" charoff="50">Cell parameters a, b, c (Å)</td><td align="center" valign="top" charoff="50">29.13, 110.52, 109.81</td><td align="center" valign="top" charoff="50">29.51, 110.49, 109.81</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">90.00, 90.61, 90.00</td><td align="center" valign="top" charoff="50">90.00, 90.74, 90.00</td></tr><tr><td colspan="3" align="left" valign="top" charoff="50"><bold>Data processing</bold></td></tr><tr><td align="left" valign="top" charoff="50">Wavelength (Å)</td><td align="center" valign="top" charoff="50">1.00000</td><td align="center" valign="top" charoff="50">0.97923</td></tr><tr><td align="left" valign="top" charoff="50">Resolution (Å)</td><td align="center" valign="top" charoff="50">50-2.60</td><td align="center" valign="top" charoff="50">50–2.90</td></tr><tr><td align="left" valign="top" charoff="50">R_merge (%)<xref ref-type="fn" rid="t1-fn1">a</xref></td><td align="center" valign="top" charoff="50">6.1 (38.7)<xref ref-type="fn" rid="t1-fn1">*</xref></td><td align="center" valign="top" charoff="50">9.4 (40.6)</td></tr><tr><td align="left" valign="top" charoff="50">I/σ</td><td align="center" valign="top" charoff="50">29.4 (4.6)</td><td align="center" valign="top" charoff="50">21.0 (3.3)</td></tr><tr><td align="left" valign="top" charoff="50">Completeness (%)</td><td align="center" valign="top" charoff="50">99.5 (99.3)</td><td align="center" valign="top" charoff="50">99.9 (100.0)</td></tr><tr><td align="left" valign="top" charoff="50">Redundancy</td><td align="center" valign="top" charoff="50">4.1 (4.1)</td><td align="center" valign="top" charoff="50">3.8 (3.8)</td></tr><tr><td align="left" valign="top" charoff="50">Measured reflections</td><td align="center" valign="top" charoff="50">88070</td><td align="center" valign="top" charoff="50">116951</td></tr><tr><td align="left" valign="top" charoff="50">Unique reflections</td><td align="center" valign="top" charoff="50">21479</td><td align="center" valign="top" charoff="50">30823</td></tr><tr><td colspan="3" align="left" valign="top" charoff="50"><bold>Refinement statistics</bold></td></tr><tr><td align="left" valign="top" charoff="50">Data range (Å)</td><td align="center" valign="top" charoff="50">30-2.60</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Reflections</td><td align="center" valign="top" charoff="50">21446</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Nonhydrogen atoms</td><td align="center" valign="top" charoff="50">5402</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Water molecules</td><td align="center" valign="top" charoff="50">47</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">R.m.s. ∆ bonds (Å)<xref ref-type="fn" rid="t1-fn2">b</xref></td><td align="center" valign="top" charoff="50">0.010</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">R.m.s. ∆ angles (°)<xref ref-type="fn" rid="t1-fn2">b</xref></td><td align="center" valign="top" charoff="50">1.365</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">R-factor (%)<xref ref-type="fn" rid="t1-fn3">c</xref></td><td align="center" valign="top" charoff="50">20.7</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">R_free (%)<xref ref-type="fn" rid="t1-fn3">c</xref>^{,<xref ref-type="fn" rid="t1-fn4">d</xref>}</td><td align="center" valign="top" charoff="50">27.7</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Ramachandran plot, residues in</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Most favored regions (%)</td><td align="center" valign="top" charoff="50">92.8</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Additional allowed regions (%)</td><td align="center" valign="top" charoff="50">6.5</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Generously allowed regions (%)</td><td align="center" valign="top" charoff="50">0.7</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Disallowed regions (%)</td><td align="center" valign="top" charoff="50">0.0</td><td align="center" valign="top" charoff="50"> </td></tr></tbody></table> 49187   SEL1Lcent   Data set: Native Se-SAD PDB accession #: 5B26   X-ray source Beamline 7A, PAL Beamline 7A, PAL Temperature (K) 100 100 Space group: P21 P21 Cell parameters a, b, c (Å) 29.13, 110.52, 109.81 29.51, 110.49, 109.81   90.00, 90.61, 90.00 90.00, 90.74, 90.00 Data processing Wavelength (Å) 1.00000 0.97923 Resolution (Å) 50-2.60 50–2.90 Rmerge (%)a 6.1 (38.7)* 9.4 (40.6) I/σ 29.4 (4.6) 21.0 (3.3) Completeness (%) 99.5 (99.3) 99.9 (100.0) Redundancy 4.1 (4.1) 3.8 (3.8) Measured reflections 88070 116951 Unique reflections 21479 30823 Refinement statistics Data range (Å) 30-2.60   Reflections 21446   Nonhydrogen atoms 5402   Water molecules 47   R.m.s. ∆ bonds (Å)b 0.010   R.m.s. ∆ angles (°)b 1.365   R-factor (%)c 20.7   Rfree (%)c,d 27.7   Ramachandran plot, residues in     Most favored regions (%) 92.8   Additional allowed regions (%) 6.5   Generously allowed regions (%) 0.7   Disallowed regions (%) 0.0   t1.xml t1 TABLE table_footnote 50207 *Highest resolution shell is shown in parenthesis. t1.xml t1 TABLE table_footnote 50258 aRmerge = 100 × ∑h∑i | Ii(h) − <I(h) >|/∑h <I(h)>, where Ii(h) is the ith measurement and <I(h)> is the weighted mean of all measurement of I(h) for Miller indices h. t1.xml t1 TABLE table_footnote 50446 bRoot-mean-squared deviation (r.m.s. ∆) from target geometries. t1.xml t1 TABLE table_footnote 50512 cR-factor = 100 × ∑|FP – FP(calc)|/∑ FP. t1.xml t1 TABLE table_footnote 50572 dRfree was calculated with 5% of the data.