PMC 20201220 pmc.key 4772114 CC BY no 0 0 10.1038/srep22324 srep22324 4772114 26927947 22324 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:Yokogawa;given-names:Mariko surname:Tsushima;given-names:Takashi surname:Noda;given-names:Nobuo N. surname:Kumeta;given-names:Hiroyuki surname:Enokizono;given-names:Yoshiaki surname:Yamashita;given-names:Kazuo surname:Standley;given-names:Daron M. surname:Takeuchi;given-names:Osamu surname:Akira;given-names:Shizuo surname:Inagaki;given-names:Fuyuhiko TITLE front 6 2016 0 Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions 0.99706316 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 ABSTRACT abstract 101 Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. 0.9963355 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 0.9934494 protein_type cleaner0 2023-07-06T12:55:04Z MESH: RNase chemical CHEBI: cleaner0 2023-07-06T12:55:18Z mRNAs protein_type MESH: cleaner0 2023-07-06T12:54:16Z IL-6 protein_type MESH: cleaner0 2023-07-06T12:54:52Z IL-12p40 0.99686384 evidence cleaner0 2023-07-06T13:52:13Z DUMMY: structures 0.9966231 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 0.99424475 species cleaner0 2023-07-06T12:55:35Z MESH: Mus musculus 0.95943165 structure_element cleaner0 2023-07-06T12:55:54Z SO: N-terminal domain 0.9985343 structure_element cleaner0 2023-07-06T12:55:57Z SO: NTD 0.9938652 structure_element cleaner0 2023-07-06T12:55:45Z SO: PilT N-terminus like 0.9969989 structure_element cleaner0 2023-07-06T12:55:49Z SO: PIN 0.9811925 structure_element cleaner0 2023-07-06T12:57:31Z SO: zinc finger 0.5748093 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.9770051 structure_element cleaner0 2023-07-06T12:57:38Z SO: C-terminal domain 0.9980526 structure_element cleaner0 2023-07-06T12:57:42Z SO: CTD 0.9985806 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase site SO: cleaner0 2023-07-06T12:58:08Z catalytic center 0.99840075 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99846005 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.99839157 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_state DUMMY: cleaner0 2023-07-06T13:03:21Z head-to-tail 0.9782215 oligomeric_state cleaner0 2023-07-06T13:11:19Z DUMMY: oligomer 0.99730396 site cleaner0 2023-07-06T13:42:58Z SO: dimer interface 0.9981868 site cleaner0 2023-07-06T14:02:46Z SO: NTD binding site 0.81668115 experimental_method cleaner0 2023-07-06T14:12:00Z MESH: mutations 0.6376089 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase structure_element SO: cleaner0 2023-07-06T12:55:58Z NTD protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.99546367 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.9505898 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.29619977 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.860189 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.8859175 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN INTRO paragraph 1084 The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. Since aberrant activation of this system leads to auto immune disorders, it must be tightly regulated. Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA. 0.9920222 protein_type cleaner0 2023-07-06T12:59:53Z MESH: pattern-recognition receptors 0.98915255 protein_type cleaner0 2023-07-06T12:59:56Z MESH: PRRs 0.94873893 protein_type cleaner0 2023-07-06T12:59:59Z MESH: Toll-like receptors 0.9913421 protein_type cleaner0 2023-07-06T13:00:04Z MESH: TLRs 0.9883321 protein_type cleaner0 2023-07-06T13:00:05Z MESH: TLRs 0.9960312 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 0.99511456 protein cleaner0 2023-07-06T12:59:45Z PR: Zc3h12a 0.9936785 protein cleaner0 2023-07-06T12:59:50Z PR: MCPIP1 0.9958835 protein_type cleaner0 2023-07-06T12:55:05Z MESH: RNase 0.9770538 chemical cleaner0 2023-07-06T13:51:16Z CHEBI: lipopolysaccharides chemical CHEBI: cleaner0 2023-07-06T12:55:19Z mRNAs protein_type MESH: cleaner0 2023-07-06T12:59:01Z (IL)-6 protein_type MESH: cleaner0 2023-07-06T12:59:18Z IL-1β protein_type MESH: cleaner0 2023-07-06T13:00:54Z IL-2 protein_type MESH: cleaner0 2023-07-06T12:54:53Z IL-12p40 0.99513847 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 chemical CHEBI: cleaner0 2023-07-06T13:00:25Z mRNA 0.9653266 structure_element cleaner0 2023-07-06T13:00:11Z SO: 3′-terminal untranslated region 0.9756991 structure_element cleaner0 2023-07-06T13:07:06Z SO: 3′UTR 0.91452265 chemical cleaner0 2023-07-06T13:00:26Z CHEBI: mRNA INTRO paragraph 1904 Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported. The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA. In addition, Regnase-1 has been predicted to possess other domains in the N- and C- terminal regions. However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved. 0.99689364 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 0.9889549 protein_type cleaner0 2023-07-06T13:51:03Z MESH: Regnase family 0.99654186 structure_element cleaner0 2023-07-06T14:05:32Z SO: PilT N-terminus like 0.99797446 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9965479 structure_element cleaner0 2023-07-06T14:05:37Z SO: CCCH-type zinc–finger 0.8693028 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.997024 protein_state cleaner0 2023-07-06T14:09:26Z DUMMY: conserved 0.95484096 protein_type cleaner0 2023-07-06T13:51:07Z MESH: Regnase family members 0.99717337 evidence cleaner0 2023-07-06T13:52:18Z DUMMY: crystal structure 0.99680424 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN 0.99518687 species cleaner0 2023-07-06T13:01:57Z MESH: Homo sapiens 0.99719834 evidence cleaner0 2023-07-06T13:52:23Z DUMMY: structure 0.98988986 residue_name cleaner0 2023-07-06T14:00:50Z SO: Asp 0.9951724 site cleaner0 2023-07-06T13:10:22Z SO: catalytic center 0.97751343 chemical cleaner0 2023-07-06T13:51:20Z CHEBI: Mg2+ protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.9969994 structure_element cleaner0 2023-07-06T14:05:40Z SO: CCCH-type ZF motifs 0.9912995 protein_type cleaner0 2023-07-06T13:51:10Z MESH: RNA-binding proteins 0.9979538 chemical cleaner0 2023-07-06T13:06:21Z CHEBI: RNA 0.99688846 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 0.8052354 structure_element cleaner0 2023-07-06T14:05:45Z SO: N- and C- terminal regions 0.8256673 evidence cleaner0 2023-07-06T13:52:27Z DUMMY: structure structure_element SO: cleaner0 2023-07-06T12:57:35Z ZF 0.9932221 structure_element cleaner0 2023-07-06T14:05:49Z SO: N-terminal domain 0.99760604 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9944104 structure_element cleaner0 2023-07-06T14:05:52Z SO: C-terminal domain 0.9967349 structure_element cleaner0 2023-07-06T12:57:43Z SO: CTD 0.9967521 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 INTRO paragraph 2729 Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro. Our data revealed that the catalytic activity of Regnase-1 is regulated through both intra and intermolecular domain interactions in vitro. The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions. Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. 0.9942131 experimental_method cleaner0 2023-07-06T14:12:08Z MESH: structural and functional analyses 0.9970216 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 0.9943695 species cleaner0 2023-07-06T12:55:36Z MESH: Mus musculus 0.99688476 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 0.99837047 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99365187 chemical cleaner0 2023-07-06T13:00:26Z CHEBI: mRNA structure_element SO: cleaner0 2023-07-06T12:55:58Z NTD structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN 0.99676126 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 0.9967153 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN 0.9953933 chemical cleaner0 2023-07-06T13:00:26Z CHEBI: mRNA 0.9967844 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 0.99598 chemical cleaner0 2023-07-06T13:00:26Z CHEBI: mRNA 0.9966903 protein_state cleaner0 2023-07-06T14:09:32Z DUMMY: NTD-activated 0.9873679 protein_state cleaner0 2023-07-06T14:09:41Z DUMMY: functional 0.97070014 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9956506 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer 0.9979144 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF chemical CHEBI: cleaner0 2023-07-06T13:06:21Z RNA 0.9399824 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9955165 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer RESULTS title_1 3441 Results RESULTS title_2 3449 Domain structures of Regnase-1 0.7206502 evidence cleaner0 2023-07-06T13:52:32Z DUMMY: structures 0.99688196 protein cleaner0 2023-07-06T12:54:59Z PR: Regnase-1 RESULTS paragraph 3480 We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. This suggests that the PIN and ZF domains exist independently without interacting with each other. The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server. 0.99682 protein cleaner0 2023-07-06T13:50:57Z PR: Rengase-1 0.9952018 species cleaner0 2023-07-06T12:55:36Z MESH: Mus musculus 0.8869559 experimental_method cleaner0 2023-07-06T14:12:12Z MESH: solved 0.99640036 evidence cleaner0 2023-07-06T13:52:36Z DUMMY: structures 0.9984811 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9984528 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9985355 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.998519 structure_element cleaner0 2023-07-06T12:57:43Z SO: CTD 0.9959334 experimental_method cleaner0 2023-07-06T13:25:25Z MESH: X-ray crystallography 0.99162745 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR 0.99591327 experimental_method cleaner0 2023-07-06T13:25:25Z MESH: X-ray crystallography 0.99827635 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99820614 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.9959991 evidence cleaner0 2023-07-06T13:52:40Z DUMMY: electron density 0.99839526 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9966171 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.99525744 species cleaner0 2023-07-06T13:01:57Z MESH: Homo sapiens 0.9983779 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9983346 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.9134344 evidence cleaner0 2023-07-06T13:52:44Z DUMMY: structures 0.99859136 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9986003 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.99869186 structure_element cleaner0 2023-07-06T12:57:43Z SO: CTD 0.9929824 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR 0.99878925 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9986803 structure_element cleaner0 2023-07-06T12:57:43Z SO: CTD structure_element SO: cleaner0 2023-07-06T14:06:11Z α helices protein PR: cleaner0 2023-07-06T13:04:43Z ubiquitin conjugating enzyme E2 K protein PR: cleaner0 2023-07-06T13:05:07Z ubiquitin associated protein 1 0.9484439 experimental_method cleaner0 2023-07-06T14:12:17Z MESH: Dali server RESULTS title_2 4303 Contribution of each domain of Regnase-1 to the mRNA binding activity 0.99704736 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA RESULTS paragraph 4373 Although the PIN domain is responsible for the catalytic activity of Regnase-1, the roles of the other domains are largely unknown. First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f). Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding. While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes. The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10 ± 1.1 μM (Supplementary Fig. 2). These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA. 0.99853945 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99647266 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.9984365 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99807453 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA 0.89889586 experimental_method cleaner0 2023-07-06T14:12:24Z MESH: in vitro gel shift assay protein_state DUMMY: cleaner0 2023-07-06T13:52:00Z Fluorescently 5′-labeled 0.9976273 chemical cleaner0 2023-07-06T13:06:21Z CHEBI: RNA protein_type MESH: cleaner0 2023-07-06T12:54:16Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA structure_element SO: cleaner0 2023-07-06T13:07:07Z 3′UTR protein_state DUMMY: cleaner0 2023-07-06T13:53:06Z inactive protein_state DUMMY: cleaner0 2023-07-06T13:19:22Z mutant 0.99841 mutant cleaner0 2023-07-06T13:07:42Z MESH: D226N 0.998334 mutant cleaner0 2023-07-06T13:07:46Z MESH: D244N 0.9962869 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 mutant MESH: cleaner0 2023-07-06T13:08:09Z DDNN protein_state DUMMY: cleaner0 2023-07-06T13:08:20Z mutant 0.9952104 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 evidence DUMMY: cleaner0 2023-07-06T14:10:14Z fluorescence 0.7604997 protein_state cleaner0 2023-07-06T14:09:52Z DUMMY: free 0.99726033 chemical cleaner0 2023-07-06T13:06:21Z CHEBI: RNA 0.99568486 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.8467543 protein_state cleaner0 2023-07-06T14:10:23Z DUMMY: bound to 0.9962359 chemical cleaner0 2023-07-06T13:06:21Z CHEBI: RNA chemical CHEBI: cleaner0 2023-07-06T13:06:21Z RNA 0.99588823 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 chemical CHEBI: cleaner0 2023-07-06T13:06:21Z RNA chemical CHEBI: cleaner0 2023-07-06T13:06:21Z RNA protein_state DUMMY: cleaner0 2023-07-06T13:06:08Z presence of 0.9979705 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD protein_state DUMMY: cleaner0 2023-07-06T13:50:31Z presence of 0.99823594 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.9982657 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.9935913 chemical cleaner0 2023-07-06T13:06:21Z CHEBI: RNA 0.9553847 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR 0.95748454 evidence cleaner0 2023-07-06T13:53:11Z DUMMY: spectral changes 0.8526306 evidence cleaner0 2023-07-06T13:53:14Z DUMMY: titration curve 0.99872357 residue_name_number cleaner0 2023-07-06T13:58:23Z DUMMY: Y314 0.98858047 evidence cleaner0 2023-07-06T13:53:16Z DUMMY: dissociation constant 0.98417723 evidence cleaner0 2023-07-06T13:28:19Z DUMMY: Kd 0.99861693 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9984654 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF chemical CHEBI: cleaner0 2023-07-06T13:06:21Z RNA 0.9986304 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9904041 chemical cleaner0 2023-07-06T13:06:21Z CHEBI: RNA RESULTS title_2 5661 Contribution of each domain of Regnase-1 to RNase activity 0.9970178 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase RESULTS paragraph 5720 In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products. The apparent half-life (T1/2) of the RNase activity was about 20 minutes. Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 > 90 minutes). It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding. protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.99303466 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.9693205 experimental_method cleaner0 2023-07-06T14:12:28Z MESH: in vitro cleavage assay protein_state DUMMY: cleaner0 2023-07-06T13:52:00Z fluorescently 5′-labeled 0.99763703 chemical cleaner0 2023-07-06T13:06:21Z CHEBI: RNA protein_type MESH: cleaner0 2023-07-06T12:54:16Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA structure_element SO: cleaner0 2023-07-06T13:07:07Z 3′UTR 0.91905814 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.8367156 mutant cleaner0 2023-07-06T13:59:49Z MESH: NTD-PIN-ZF 0.7324506 chemical cleaner0 2023-07-06T13:00:26Z CHEBI: mRNA protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.9866276 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.98778534 protein_state cleaner0 2023-07-06T14:10:28Z DUMMY: lacking structure_element SO: cleaner0 2023-07-06T12:57:35Z ZF 0.9885648 protein_state cleaner0 2023-07-06T14:10:31Z DUMMY: lacking 0.9975284 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9904258 mutant cleaner0 2023-07-06T13:59:52Z MESH: NTD-PIN(DDNN)-ZF 0.99837947 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.88263583 protein_state cleaner0 2023-07-06T14:10:34Z DUMMY: lacks site SO: cleaner0 2023-07-06T14:03:13Z catalytic residues 0.99856585 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase site SO: cleaner0 2023-07-06T13:10:21Z catalytic center 0.99861443 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99850696 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.9938163 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA RESULTS title_2 6810 Dimer formation of the PIN domains oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:23Z Dimer structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN RESULTS paragraph 6845 During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a). By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20–200 μM range. The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively. We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3). Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure. Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). The side chains of these residues point away from the catalytic center on the same molecule (Fig. 2b). Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro. 0.49429753 experimental_method cleaner0 2023-07-06T14:12:40Z MESH: purification 0.98449016 experimental_method cleaner0 2023-07-06T14:12:42Z MESH: gel filtration 0.9975926 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN experimental_method MESH: cleaner0 2023-07-06T13:53:47Z comparison with the elution volume of standard marker proteins 0.9975043 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9934463 oligomeric_state cleaner0 2023-07-06T13:11:28Z DUMMY: monomer 0.99505746 oligomeric_state cleaner0 2023-07-06T13:11:22Z DUMMY: dimer 0.9972782 evidence cleaner0 2023-07-06T13:53:51Z DUMMY: crystal structure structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN 0.9903691 evidence cleaner0 2023-07-06T13:53:56Z DUMMY: crystal forms 0.99769187 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.7567082 protein_state cleaner0 2023-07-06T13:11:11Z DUMMY: head-to-tail 0.61930215 oligomeric_state cleaner0 2023-07-06T13:11:19Z DUMMY: oligomer 0.99009776 evidence cleaner0 2023-07-06T13:53:59Z DUMMY: crystal forms 0.97776425 experimental_method cleaner0 2023-07-06T14:12:48Z MESH: Mutation 0.9985078 residue_name_number cleaner0 2023-07-06T13:58:29Z DUMMY: Arg215 site SO: cleaner0 2023-07-06T14:01:25Z oligomeric surface 0.9858667 residue_name cleaner0 2023-07-06T14:00:56Z SO: Glu oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:29Z monomer oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:23Z dimer 0.99677914 protein_state cleaner0 2023-07-06T13:10:58Z DUMMY: wild type 0.9691986 experimental_method cleaner0 2023-07-06T14:12:51Z MESH: single mutations structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:29Z monomer 0.95763874 experimental_method cleaner0 2023-07-06T14:12:58Z MESH: gel filtration 0.9966627 protein_state cleaner0 2023-07-06T13:10:58Z DUMMY: Wild type 0.81764865 oligomeric_state cleaner0 2023-07-06T13:54:29Z DUMMY: monomeric structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants 0.9986314 mutant cleaner0 2023-07-06T13:11:34Z MESH: P212A 0.9985985 mutant cleaner0 2023-07-06T13:11:39Z MESH: D278R 0.99153614 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR 0.9876031 evidence cleaner0 2023-07-06T13:54:02Z DUMMY: spectra 0.996472 site cleaner0 2023-07-06T13:42:59Z SO: dimer interface 0.99663275 protein_state cleaner0 2023-07-06T13:10:58Z DUMMY: wild type 0.9971969 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.77656555 oligomeric_state cleaner0 2023-07-06T13:54:30Z DUMMY: monomeric protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants 0.9976539 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.8635138 protein_state cleaner0 2023-07-06T13:11:11Z DUMMY: head-to-tail 0.95340234 oligomeric_state cleaner0 2023-07-06T13:11:18Z DUMMY: oligomer 0.9970512 evidence cleaner0 2023-07-06T13:54:06Z DUMMY: crystal structure 0.6994014 oligomeric_state cleaner0 2023-07-06T13:54:30Z DUMMY: monomeric 0.3403942 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants 0.99864393 mutant cleaner0 2023-07-06T13:11:35Z MESH: P212A 0.99861157 mutant cleaner0 2023-07-06T13:11:44Z MESH: R214A 0.99861526 mutant cleaner0 2023-07-06T13:11:40Z MESH: D278R protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase protein_type MESH: cleaner0 2023-07-06T12:54:16Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA 0.77138484 site cleaner0 2023-07-06T13:10:22Z SO: catalytic center protein_state DUMMY: cleaner0 2023-07-06T13:11:11Z head-to-tail 0.487095 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99777055 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9731679 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase RESULTS title_2 8692 Domain-domain interaction between the NTD and the PIN domain 0.9979849 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99878246 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN RESULTS paragraph 8753 While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h). In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). These results clearly indicate a direct interaction between the PIN domain and the NTD. Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c). 0.9982394 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD chemical CHEBI: cleaner0 2023-07-06T13:06:21Z RNA protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.99550647 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.64479476 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9949989 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.9981713 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99805367 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.984757 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR 0.9969028 protein_state cleaner0 2023-07-06T14:10:39Z DUMMY: catalytically inactive 0.55651623 oligomeric_state cleaner0 2023-07-06T13:54:30Z DUMMY: monomeric structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN protein_state DUMMY: cleaner0 2023-07-06T13:13:23Z mutant 0.9971111 mutant cleaner0 2023-07-06T13:08:10Z MESH: DDNN 0.99854714 mutant cleaner0 2023-07-06T13:11:40Z MESH: D278R oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:23Z dimer 0.99785584 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.6698075 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR 0.99814427 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99865305 residue_name_number cleaner0 2023-07-06T13:58:33Z DUMMY: V177 residue_range DUMMY: cleaner0 2023-07-06T13:13:50Z F210-T211 0.9985494 residue_name_number cleaner0 2023-07-06T13:47:11Z DUMMY: R214 residue_range DUMMY: cleaner0 2023-07-06T13:14:07Z F228-L232 residue_range DUMMY: cleaner0 2023-07-06T13:14:27Z F234-S236 experimental_method MESH: cleaner0 2023-07-06T14:13:26Z addition of 0.9972126 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.63193554 experimental_method cleaner0 2023-07-06T14:13:30Z MESH: addition of 0.9901776 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN experimental_method MESH: cleaner0 2023-07-06T13:24:17Z NMR 0.99856794 residue_name_number cleaner0 2023-07-06T13:58:37Z DUMMY: R56 residue_range DUMMY: cleaner0 2023-07-06T13:14:43Z L58-G59 residue_range DUMMY: cleaner0 2023-07-06T13:15:01Z V86-H88 0.9978836 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99863356 residue_name_number cleaner0 2023-07-06T13:58:41Z DUMMY: D53 0.99868435 residue_name_number cleaner0 2023-07-06T13:58:44Z DUMMY: F55 0.998694 residue_name_number cleaner0 2023-07-06T13:58:46Z DUMMY: K57 residue_range DUMMY: cleaner0 2023-07-06T13:15:18Z Y60-S61 0.9986437 residue_name_number cleaner0 2023-07-06T13:58:50Z DUMMY: V68 residue_range DUMMY: cleaner0 2023-07-06T13:15:36Z T80-G83 0.9986634 residue_name_number cleaner0 2023-07-06T13:58:53Z DUMMY: L85 0.9986873 residue_name_number cleaner0 2023-07-06T13:58:56Z DUMMY: G89 0.9984981 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99816114 residue_name_number cleaner0 2023-07-06T13:58:59Z DUMMY: N79 0.9981133 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9982187 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.93074256 evidence cleaner0 2023-07-06T13:54:52Z DUMMY: titration curve evidence DUMMY: cleaner0 2023-07-06T13:55:10Z chemical shift changes 0.9986505 residue_name_number cleaner0 2023-07-06T13:59:02Z DUMMY: L58 0.99329054 evidence cleaner0 2023-07-06T13:28:19Z DUMMY: Kd 0.99828786 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.96001387 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9982845 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99725014 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9599482 structure_element cleaner0 2023-07-06T14:06:22Z SO: linker 0.98603517 evidence cleaner0 2023-07-06T13:55:14Z DUMMY: binding affinity 0.6444536 protein_state cleaner0 2023-07-06T14:10:42Z DUMMY: native 0.9977081 site cleaner0 2023-07-06T14:03:19Z SO: PIN/NTD interface 0.97318345 structure_element cleaner0 2023-07-06T14:06:27Z SO: helix 0.9988331 residue_name_number cleaner0 2023-07-06T13:59:06Z DUMMY: D225 0.9988852 residue_name_number cleaner0 2023-07-06T13:59:09Z DUMMY: D226 0.9984113 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9964691 site cleaner0 2023-07-06T14:03:23Z SO: binding site 0.9971938 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99723375 site cleaner0 2023-07-06T13:44:44Z SO: PIN-PIN dimer interface structure_element SO: cleaner0 2023-07-06T12:55:58Z NTD structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN 0.68101674 experimental_method cleaner0 2023-07-06T14:13:33Z MESH: in silico docking 0.9981969 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99754286 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9163096 evidence cleaner0 2023-07-06T13:55:17Z DUMMY: chemical shift restraints 0.9760098 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR RESULTS title_2 10692 Residues critical for Regnase-1 RNase activity 0.9926675 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase RESULTS paragraph 10739 To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains. 0.9933803 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.9982642 site cleaner0 2023-07-06T13:17:44Z SO: catalytic site 0.5471427 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.85306406 oligomeric_state cleaner0 2023-07-06T13:11:19Z DUMMY: oligomer 0.95547485 experimental_method cleaner0 2023-07-06T14:13:37Z MESH: mutated to 0.98409975 residue_name cleaner0 2023-07-06T14:01:44Z SO: alanine protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.99340606 experimental_method cleaner0 2023-07-06T14:13:41Z MESH: gel filtration assays protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants 0.99769235 mutant cleaner0 2023-07-06T13:11:44Z MESH: R214A 0.9367153 oligomeric_state cleaner0 2023-07-06T14:02:14Z DUMMY: dimers protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants 0.9975062 mutant cleaner0 2023-07-06T13:11:44Z MESH: R214A oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:23Z dimer 0.99367803 mutant cleaner0 2023-07-06T13:21:17Z MESH: W182A 0.9938613 mutant cleaner0 2023-07-06T13:59:57Z MESH: R183A 0.9961654 mutant cleaner0 2023-07-06T13:11:44Z MESH: R214A protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants protein_type MESH: cleaner0 2023-07-06T12:54:16Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA protein PR: cleaner0 2023-07-06T12:55:00Z Regnase-1 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA 0.9929792 mutant cleaner0 2023-07-06T13:21:22Z MESH: K184A 0.9969901 mutant cleaner0 2023-07-06T14:00:01Z MESH: R215A 0.996974 mutant cleaner0 2023-07-06T13:21:32Z MESH: R220A protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants chemical CHEBI: cleaner0 2023-07-06T12:55:19Z mRNAs 0.9987538 residue_name_number cleaner0 2023-07-06T13:46:04Z DUMMY: K219 0.99883634 residue_name_number cleaner0 2023-07-06T13:46:09Z DUMMY: R247 protein_type MESH: cleaner0 2023-07-06T12:54:16Z IL-6 0.7909548 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA 0.99872726 residue_name_number cleaner0 2023-07-06T13:46:03Z DUMMY: K219 0.9987649 residue_name_number cleaner0 2023-07-06T13:46:09Z DUMMY: R247 protein_type MESH: cleaner0 2023-07-06T12:54:16Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA 0.73296374 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA 0.998792 residue_name_number cleaner0 2023-07-06T13:46:13Z DUMMY: K152 0.99888474 residue_name_number cleaner0 2023-07-06T13:46:17Z DUMMY: R158 0.9988695 residue_name_number cleaner0 2023-07-06T13:46:22Z DUMMY: R188 0.9988493 residue_name_number cleaner0 2023-07-06T13:46:26Z DUMMY: R200 0.99886715 residue_name_number cleaner0 2023-07-06T13:46:30Z DUMMY: K204 0.9988532 residue_name_number cleaner0 2023-07-06T13:46:35Z DUMMY: K206 0.99883693 residue_name_number cleaner0 2023-07-06T13:46:43Z DUMMY: K257 0.99881834 residue_name_number cleaner0 2023-07-06T13:46:49Z DUMMY: R258 protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.99880743 residue_name_number cleaner0 2023-07-06T13:46:54Z DUMMY: W182 0.9988092 residue_name_number cleaner0 2023-07-06T13:46:59Z DUMMY: R183 0.7364476 oligomeric_state cleaner0 2023-07-06T13:54:30Z DUMMY: monomeric 0.41677222 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9933302 evidence cleaner0 2023-07-06T13:55:23Z DUMMY: structure protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase site SO: cleaner0 2023-07-06T13:17:44Z catalytic site 0.9987658 residue_name_number cleaner0 2023-07-06T13:47:03Z DUMMY: K184 0.99793285 site cleaner0 2023-07-06T14:03:30Z SO: active site 0.71547395 evidence cleaner0 2023-07-06T13:55:28Z DUMMY: structure 0.998803 residue_name_number cleaner0 2023-07-06T13:47:04Z DUMMY: K184 0.6941805 protein_state cleaner0 2023-07-06T13:45:37Z DUMMY: primary” 0.9955155 site cleaner0 2023-07-06T13:17:44Z SO: catalytic site 0.9988438 residue_name_number cleaner0 2023-07-06T13:47:11Z DUMMY: R214 0.9961128 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99882454 residue_name_number cleaner0 2023-07-06T13:47:11Z DUMMY: R214 0.632672 protein_state cleaner0 2023-07-06T13:45:54Z DUMMY: primary 0.99702793 site cleaner0 2023-07-06T14:03:34Z SO: active site 0.9939698 site cleaner0 2023-07-06T13:42:59Z SO: dimer interface 0.99103165 site cleaner0 2023-07-06T14:03:40Z SO: putative-RNA binding residues 0.99874157 residue_name_number cleaner0 2023-07-06T13:47:04Z DUMMY: K184 0.9988047 residue_name_number cleaner0 2023-07-06T13:47:11Z DUMMY: R214 0.9960449 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.6059957 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN RESULTS title_2 12527 Molecular mechanism of target mRNA cleavage by the PIN dimer chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA 0.8507944 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.98532355 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer RESULTS paragraph 12588 Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5). One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4). These were paired with a DDNN mutant that had no RNase activity by itself. When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products. This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. Taken together, the rescue experiments above support the proposed model in which the head-to-tail dimer is functional in vitro. 0.9658383 experimental_method cleaner0 2023-07-06T14:13:46Z MESH: mutational experiments 0.99637294 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer 0.973656 protein_state cleaner0 2023-07-06T14:06:42Z DUMMY: secondary 0.5243988 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.8478697 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.99665636 site cleaner0 2023-07-06T14:03:46Z SO: RNA binding residues 0.99816585 site cleaner0 2023-07-06T14:03:50Z SO: active site 0.9636926 protein_state cleaner0 2023-07-06T14:06:52Z DUMMY: primary 0.54169255 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9962474 protein_state cleaner0 2023-07-06T14:10:48Z DUMMY: catalytically inactive 0.9974141 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9963982 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.97004324 protein_state cleaner0 2023-07-06T14:10:52Z DUMMY: lacking 0.9961864 site cleaner0 2023-07-06T14:03:53Z SO: RNA-binding residues 0.99208504 protein_state cleaner0 2023-07-06T14:10:59Z DUMMY: inactive 0.9081038 protein_state cleaner0 2023-07-06T14:11:02Z DUMMY: active 0.9344517 experimental_method cleaner0 2023-07-06T14:13:50Z MESH: in vitro cleavage assays 0.974854 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.49429753 protein_state cleaner0 2023-07-06T13:20:00Z DUMMY: mutants protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.99670315 protein_state cleaner0 2023-07-06T14:11:05Z DUMMY: catalytically active structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN 0.7770609 experimental_method cleaner0 2023-07-06T14:13:56Z MESH: mutation of protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase mutant MESH: cleaner0 2023-07-06T13:08:10Z DDNN protein_state DUMMY: cleaner0 2023-07-06T13:19:22Z mutant protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.9951403 oligomeric_state cleaner0 2023-07-06T13:19:45Z DUMMY: heterodimers 0.7401305 mutant cleaner0 2023-07-06T13:08:10Z MESH: DDNN 0.9636169 protein_state cleaner0 2023-07-06T13:20:29Z DUMMY: primary 0.7979454 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_state DUMMY: cleaner0 2023-07-06T13:19:22Z mutant 0.96400183 protein_state cleaner0 2023-07-06T13:20:47Z DUMMY: secondary 0.5961947 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase protein_state DUMMY: cleaner0 2023-07-06T13:19:22Z mutant 0.8813331 protein_state cleaner0 2023-07-06T14:07:07Z DUMMY: primary 0.96059597 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.63430876 mutant cleaner0 2023-07-06T13:08:10Z MESH: DDNN 0.97392976 protein_state cleaner0 2023-07-06T14:07:17Z DUMMY: secondary 0.6291083 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase evidence DUMMY: cleaner0 2023-07-06T13:42:11Z fluorescence intensity protein_state DUMMY: cleaner0 2023-07-06T13:49:26Z uncleaved protein_type MESH: cleaner0 2023-07-06T12:54:16Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants 0.9852212 mutant cleaner0 2023-07-06T13:21:17Z MESH: W182A 0.9861892 mutant cleaner0 2023-07-06T13:21:22Z MESH: K184A 0.96760845 mutant cleaner0 2023-07-06T13:11:45Z MESH: R214A 0.96821 mutant cleaner0 2023-07-06T13:21:31Z MESH: R220A mutant MESH: cleaner0 2023-07-06T13:08:10Z DDNN protein_state DUMMY: cleaner0 2023-07-06T13:19:22Z mutant evidence DUMMY: cleaner0 2023-07-06T13:42:11Z fluorescence intensity protein_state DUMMY: cleaner0 2023-07-06T13:49:26Z uncleaved protein PR: cleaner0 2023-07-06T12:55:00Z Regnase-1 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants 0.9902086 mutant cleaner0 2023-07-06T13:21:22Z MESH: K184A 0.9869365 mutant cleaner0 2023-07-06T13:11:45Z MESH: R214A mutant MESH: cleaner0 2023-07-06T13:08:10Z DDNN protein_state DUMMY: cleaner0 2023-07-06T13:19:22Z mutant 0.9826202 mutant cleaner0 2023-07-06T13:21:22Z MESH: K184A 0.98075163 mutant cleaner0 2023-07-06T13:11:45Z MESH: R214A mutant MESH: cleaner0 2023-07-06T13:08:10Z DDNN protein_state DUMMY: cleaner0 2023-07-06T13:19:22Z mutant 0.9985215 residue_name_number cleaner0 2023-07-06T13:47:04Z DUMMY: K184 0.99844944 residue_name_number cleaner0 2023-07-06T13:47:11Z DUMMY: R214 protein_state DUMMY: cleaner0 2023-07-06T13:22:41Z primary structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN site SO: cleaner0 2023-07-06T13:10:22Z catalytic center protein_state DUMMY: cleaner0 2023-07-06T13:23:10Z secondary structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN site SO: cleaner0 2023-07-06T13:10:22Z catalytic center 0.8445035 protein_state cleaner0 2023-07-06T13:22:53Z DUMMY: primary 0.7418916 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9984372 residue_name_number cleaner0 2023-07-06T13:47:11Z DUMMY: R214 oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:23Z dimer 0.96471846 mutant cleaner0 2023-07-06T13:11:45Z MESH: R214A protein_state DUMMY: cleaner0 2023-07-06T13:23:31Z secondary structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN 0.996743 mutant cleaner0 2023-07-06T13:11:45Z MESH: R214A 0.9974546 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99503005 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer 0.8482282 mutant cleaner0 2023-07-06T13:08:10Z MESH: DDNN 0.99564797 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_state DUMMY: cleaner0 2023-07-06T13:23:48Z secondary structure_element SO: cleaner0 2023-07-06T12:55:50Z PIN 0.98570395 protein_state cleaner0 2023-07-06T13:11:11Z DUMMY: head-to-tail 0.99637955 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer DISCUSS title_1 14859 Discussion DISCUSS paragraph 14870 We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography. Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains. A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. These results indicate that Regnase-1 directly binds to RNA and precipitates under such experimental conditions. Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR. 0.97923684 evidence cleaner0 2023-07-06T13:55:34Z DUMMY: structures 0.99567956 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.9941907 experimental_method cleaner0 2023-07-06T13:24:16Z MESH: NMR 0.99620944 experimental_method cleaner0 2023-07-06T13:25:25Z MESH: X-ray crystallography 0.9986823 structure_element cleaner0 2023-07-06T12:57:43Z SO: CTD 0.9985928 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9983541 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99825305 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.98885316 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.9979913 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9980254 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.99311554 species cleaner0 2023-07-06T12:55:36Z MESH: Mus musculus 0.97050124 experimental_method cleaner0 2023-07-06T14:14:04Z MESH: crystallized 0.99573576 evidence cleaner0 2023-07-06T13:55:38Z DUMMY: electron density 0.9982503 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.99840987 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.9916284 protein_state cleaner0 2023-07-06T14:11:16Z DUMMY: highly mobile 0.9928031 protein_state cleaner0 2023-07-06T13:49:31Z DUMMY: absence of 0.9949568 chemical cleaner0 2023-07-06T13:00:26Z CHEBI: mRNA 0.9919566 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR 0.9981407 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.98258847 protein_type cleaner0 2023-07-06T12:54:16Z MESH: IL-6 0.93520975 chemical cleaner0 2023-07-06T13:00:26Z CHEBI: mRNA 0.99338335 evidence cleaner0 2023-07-06T13:28:19Z DUMMY: Kd 0.9251319 experimental_method cleaner0 2023-07-06T14:14:07Z MESH: in vitro gel shift assay 0.99186295 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.9976367 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA 0.9452816 chemical cleaner0 2023-07-06T13:06:22Z CHEBI: RNA 0.9949519 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.9977245 chemical cleaner0 2023-07-06T13:06:22Z CHEBI: RNA experimental_method MESH: cleaner0 2023-07-06T13:25:57Z structural analyses complex_assembly GO: cleaner0 2023-07-06T13:25:13Z mRNA-Regnase-1 0.996264 experimental_method cleaner0 2023-07-06T13:25:24Z MESH: X-ray crystallography 0.99421906 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR DISCUSS paragraph 15945 The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments. In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. This inconsistency might be due to difference in the analytical methods and/or protein concentrations used in each experiment, since the oligomer formation of PIN was dependent on the protein concentration in our study. 0.997193 evidence cleaner0 2023-07-06T13:55:41Z DUMMY: crystal structure 0.9956177 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.9980925 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9952601 species cleaner0 2023-07-06T13:01:57Z MESH: Homo sapiens 0.9953058 species cleaner0 2023-07-06T12:55:36Z MESH: Mus musculus 0.9115232 evidence cleaner0 2023-07-06T13:55:45Z DUMMY: RMSD 0.90184754 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.98497796 residue_range cleaner0 2023-07-06T13:47:29Z DUMMY: 134–295 0.9944193 taxonomy_domain cleaner0 2023-07-06T13:26:14Z DUMMY: mouse 0.9957675 species cleaner0 2023-07-06T13:26:21Z MESH: human 0.99777824 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.97708714 protein_state cleaner0 2023-07-06T13:11:11Z DUMMY: head-to-tail 0.98037237 oligomeric_state cleaner0 2023-07-06T14:02:19Z DUMMY: oligomers 0.99634933 evidence cleaner0 2023-07-06T13:55:48Z DUMMY: crystal forms 0.92157084 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.98985136 oligomeric_state cleaner0 2023-07-06T14:02:25Z DUMMY: monomers 0.9923066 experimental_method cleaner0 2023-07-06T14:14:12Z MESH: analytical ultra-centrifugation 0.9894432 experimental_method cleaner0 2023-07-06T14:14:16Z MESH: gel filtration 0.98712045 experimental_method cleaner0 2023-07-06T14:14:19Z MESH: mutational analyses 0.98363185 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR 0.49351272 evidence cleaner0 2023-07-06T13:55:50Z DUMMY: spectra 0.99800354 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99093944 protein_state cleaner0 2023-07-06T13:11:11Z DUMMY: head-to-tail 0.9965288 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer 0.997293 evidence cleaner0 2023-07-06T13:55:52Z DUMMY: crystal structure oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:19Z oligomer 0.67573106 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN DISCUSS paragraph 17024 Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro. 0.97857404 experimental_method cleaner0 2023-07-06T14:14:26Z MESH: Single mutations 0.8207907 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.8227865 oligomeric_state cleaner0 2023-07-06T14:02:28Z DUMMY: monomerized protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.850628 experimental_method cleaner0 2023-07-06T13:24:17Z MESH: NMR 0.7450116 evidence cleaner0 2023-07-06T13:55:58Z DUMMY: spectra 0.5644939 oligomeric_state cleaner0 2023-07-06T13:54:30Z DUMMY: monomeric protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants 0.6800348 oligomeric_state cleaner0 2023-07-06T13:54:30Z DUMMY: monomeric protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants structure_element SO: cleaner0 2023-07-06T13:48:07Z PIN structure_element SO: cleaner0 2023-07-06T13:48:15Z PIN oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:23Z dimer 0.7938333 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.99724627 evidence cleaner0 2023-07-06T13:56:01Z DUMMY: crystal structure 0.72638947 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99680114 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer protein PR: cleaner0 2023-07-06T12:55:00Z Regnase-1 protein_state DUMMY: cleaner0 2023-07-06T14:07:47Z primary protein_state DUMMY: cleaner0 2023-07-06T14:07:58Z secondary 0.72285646 structure_element cleaner0 2023-07-06T14:08:01Z SO: PINs 0.9981303 site cleaner0 2023-07-06T13:17:44Z SO: catalytic site 0.7775953 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99617386 experimental_method cleaner0 2023-07-06T14:14:29Z MESH: structure-based mutational analyses protein PR: cleaner0 2023-07-06T12:55:00Z Regnase-1 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA DISCUSS paragraph 17888 The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage. Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). Taken together, this suggests that the NTD and the PIN domain compete for a common binding site. The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo. This result is consistent with a model in which the NTD acts as an enhancer, and cleavage of the linker lowers enzymatic activity dramatically. 0.9923765 experimental_method cleaner0 2023-07-06T14:14:33Z MESH: cleavage assay 0.998659 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.6335911 chemical cleaner0 2023-07-06T13:00:26Z CHEBI: mRNA 0.9985391 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9485144 site cleaner0 2023-07-06T14:04:02Z SO: oligomeric surface protein_state DUMMY: cleaner0 2023-07-06T14:08:46Z primary 0.99635243 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.98555815 structure_element cleaner0 2023-07-06T14:08:05Z SO: helix 0.7927509 site cleaner0 2023-07-06T14:04:06Z SO: catalytic residues 0.9986872 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99848086 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.96827227 site cleaner0 2023-07-06T14:04:27Z SO: common binding site 0.8903143 evidence cleaner0 2023-07-06T13:56:04Z DUMMY: affinity 0.9919689 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN evidence DUMMY: cleaner0 2023-07-06T13:28:19Z Kd 0.81601435 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.6422894 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN evidence DUMMY: cleaner0 2023-07-06T13:28:19Z Kd 0.97889966 residue_range cleaner0 2023-07-06T14:00:35Z DUMMY: 90–133 0.99872094 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD protein_state DUMMY: cleaner0 2023-07-06T14:08:33Z primary 0.99739 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99738455 protein_state cleaner0 2023-07-06T14:11:22Z DUMMY: full-length 0.9968293 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.95738995 experimental_method cleaner0 2023-07-06T14:14:36Z MESH: docking and molecular dynamics simulations structure_element SO: cleaner0 2023-07-06T12:55:58Z NTD 0.6960801 site cleaner0 2023-07-06T14:04:33Z SO: catalytic residues 0.99850863 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99502134 protein_state cleaner0 2023-07-06T14:11:27Z DUMMY: active 0.99603933 chemical cleaner0 2023-07-06T13:52:07Z CHEBI: Mg2+ 0.998787 protein cleaner0 2023-07-06T13:29:05Z PR: Malt1 0.99686813 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.99852484 residue_name_number cleaner0 2023-07-06T13:59:16Z DUMMY: R111 0.99869967 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.998374 structure_element cleaner0 2023-07-06T14:08:09Z SO: linker DISCUSS paragraph 19145 Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b). The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1. 0.9916974 experimental_method cleaner0 2023-07-06T14:14:41Z MESH: structural and functional analyses 0.99671763 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.99478006 experimental_method cleaner0 2023-07-06T14:14:44Z MESH: docking simulations 0.64951575 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.7890433 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9949609 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer 0.9901457 protein_type cleaner0 2023-07-06T12:54:16Z MESH: IL-6 0.976857 chemical cleaner0 2023-07-06T13:00:26Z CHEBI: mRNA 0.98416615 site cleaner0 2023-07-06T14:04:37Z SO: cleavage site protein_type MESH: cleaner0 2023-07-06T12:54:16Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:26Z mRNA 0.97995067 experimental_method cleaner0 2023-07-06T14:14:48Z MESH: polyacrylamide gel electrophoresis 0.90254337 experimental_method cleaner0 2023-07-06T14:14:51Z MESH: docking chemical CHEBI: cleaner0 2023-07-06T13:06:22Z RNA protein_type MESH: cleaner0 2023-07-06T13:30:27Z RNA-binding proteins 0.74459183 structure_element cleaner0 2023-07-06T14:09:02Z SO: loop 0.99668485 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 DISCUSS paragraph 19757 The overall model of regulation of Regnase-1 RNase activity through domain-domain interactions in vitro is summarized in Fig. 6. In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses. 0.99697834 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:55:05Z RNase 0.994583 protein_state cleaner0 2023-07-06T13:49:31Z DUMMY: absence of 0.9962379 chemical cleaner0 2023-07-06T13:00:26Z CHEBI: mRNA 0.9986432 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.9353172 protein_state cleaner0 2023-07-06T13:11:11Z DUMMY: head-to-tail 0.99412054 oligomeric_state cleaner0 2023-07-06T14:02:33Z DUMMY: oligomers 0.9860203 protein_state cleaner0 2023-07-06T14:11:32Z DUMMY: fully active 0.9961766 site cleaner0 2023-07-06T13:10:22Z SO: catalytic center 0.9985299 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:19Z oligomer 0.9985869 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_state DUMMY: cleaner0 2023-07-06T13:11:11Z head-to-tail oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:19Z oligomer protein_state DUMMY: cleaner0 2023-07-06T13:48:38Z primary 0.95147157 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.89128834 protein_state cleaner0 2023-07-06T14:11:38Z DUMMY: functional 0.9970004 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer 0.9982644 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN protein_state DUMMY: cleaner0 2023-07-06T13:48:49Z secondary 0.9390025 structure_element cleaner0 2023-07-06T12:55:50Z SO: PIN 0.99683267 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.99686545 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein PR: cleaner0 2023-07-06T13:31:15Z Regnase-1 METHODS title_1 20555 Methods METHODS title_2 20563 Protein expression and purification METHODS paragraph 20599 The DNA fragment encoding Regnase-1 derived from Mus musculus was cloned into pGEX6p vector (GE Healthcare). All the mutants were generated by PCR-mediated site-directed mutagenesis and confirmed by the DNA sequence analyses. As a catalytically deficient mutant, both Asp226 and Asp244 at the catalytic center of PIN were mutated to Asn, which is referred to as DDNN mutant. Regnase-1 was expressed at 16 °C using the Escherichia coli RosettaTM(DE3)pLysS strain. After purification with a GST-affinity resin, an N-terminal GST tag was digested by HRV-3 C protease. NTD was further purified by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). The other domains were further purified by cation exchange chromatography using Resource S (GE Healthcare), followed by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). Uniformly 15N or 13C, 15N-double labeled proteins for NMR experiments were prepared by growing E. coli host in M9 minimal medium containing 15NH4Cl, unlabeled glucose and 15N CELTONE® Base Powder (CIL) or 15NH4Cl, 13C6-glucose, and13C, 15N CELTONE® Base Powder (CIL), respectively. METHODS title_2 21771 X-ray crystallography METHODS paragraph 21793 Crystallization was performed using the sitting drop vapor diffusion method at 20 °C and two crystal forms (I and II) were obtained. In the case of form I crystals, drops (0.5 μl) of 6 mg/ml selenomethionine-labeled Regnase-1 PIN-ZF (residues 134–339 derived from Mus musculus) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1 M (NH4)2HPO4, 200 mM NaCl and 100 mM sodium citrate (pH 5.5) whereas in the case of form II crystals, drops (0.5 μl) of 6 mg/ml native Regnase-1 PIN-ZF (residues 134–339) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1.7 M NaCl and 100 mM HEPES-NaOH (pH 7.0). Diffraction data were collected at a Photon Factory Advanced Ring beamline NE3A (form I) or at a SPring-8 beamline BL41XU (form II), and were processed with HKL2000. The structure of the form I crystal was determined by the multiple anomalous dispersion (MAD) method. Nine Se sites were found using the program SOLVE; however, the electron density obtained by MAD phases calculated using SOLVE was not good enough to build a model even after density modification using the program RESOLVE. Then the program CNS was used to find additional three Se sites and calculate MAD phases using 12 Se sites. The electron density after density modification using CNS was good enough to build a model. Structure of the form II crystal was determined by the molecular replacement method using CNS and using the structure of the form I crystal as a search model. For all structures, further model building was performed manually with COOT, and TLS and restrained refinement using isotropic individual B factors was performed with REFMAC5 in the CCP4 program suite. Crystallographic parameters are summarized in Supplementary Table 1. METHODS title_2 23654 NMR measurements METHODS paragraph 23671 All NMR experiments were carried out at 298 K on Inova 500-MHz, 600-MHz, and 800-MHz spectrometer (Agilent). The NMR data were processed using the NMRPipe, the Olivia (fermi.pharm.hokudai.ac.jp/olivia/), and the Sparky program (Sparky3, University of California, San Francisco). METHODS paragraph 23952 For structure calculation, NOE distance restraints were obtained from 3D 15N-NOESY-HSQC (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD) and 13C-NOESY-HSQC spectra (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD). The NMR structures were determined using the CANDID/CYANA2.1. Dihedral restraints were derived from backbone chemical shifts using TALOS. METHODS paragraph 24378 For the domain-domain interaction analyses between the NTD and the PIN domain, 1H-15N HSQC spectra of uniformly 15N-labeled proteins in the concentration of 100 μM were obtained in the presence of 3 or 6 molar equivalents of unlabeled proteins. METHODS title_2 24626 Preparation of RNAs METHODS paragraph 24646 The fluorescently labeled RNAs at the 5′-end by 6-FAM were purchased from SIGMA-ALDORICH. The RNA sequences used in this study were shown below. METHODS paragraph 24793 IL-6 mRNA 3′UTR (82–106): 5′-UGUUGUUCUCUACGAAGAACUGACA-3′ (25 nts) METHODS paragraph 24868 Regnase-1 mRNA 3′UTR (191–211): 5′- CUGUUGAUACACAUUGUAUCU-3′ (21 nts) METHODS title_2 24946 Electrophoretic mobility shift assay METHODS paragraph 24983 Catalytically deficient Regnase-1 proteins, containing DDNN mutations, and 5′-terminally 6-FAM labeled RNAs were incubated in the RNA-binding buffer (20 mM HEPES-NaOH (pH 6.8), 150 mM NaCl, 1 mM DTT, 10% glycerol (v/v), and 0.1% NP-40 (v/v)) at 4 °C for 30 minutes, then analyzed by non-denaturing polyacrylamide gel electrophoresis. The electrophoreses were performed at 4 °C using the 7.5% polyacrylamide (w/v) gel (monomer : bis = 29 : 1) in the electrophoresis buffer (25 mM Tris-HCl (pH 7.5) and 200 mM glycine). The fluorescence of 6-FAM labeled RNA was directly detected at the excitation wavelength of 460 nm with a fluorescence filter (Y515-Di) using a fluoroimaging analyzer (LAS-4000 (FUJIFILM)). The fluorescence intensity of each sample was quantified using ImageJ software. METHODS title_2 25797 In vitro RNA cleavage assay METHODS paragraph 25825 Regnase-1 (2 μM) and 5′-terminally 6-FAM labeled RNA (1 μM) were incubated in the RNA-cleavage buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 1 mM DTT) at 37 °C. For the assay using combinations of Regnase-1 mutants, equimolar amounts of Regnase-1 mutants (2 μM each) were mixed with fluorescently labeled RNA (1 μM). After incubation for 30–120 minutes, the reaction was stopped by the addition of 1.5-fold volume of denaturing buffer containing 8 M urea and 100 mM EDTA, and samples were boiled. The electrophoreses were performed at room temperature using the 8 M urea containing denaturing gel with 20% polyacrylamide (w/v) (monomer : bis = 19 : 1) in 0.5 × TBE as the electrophoresis buffer. METHODS title_2 26581 Docking calculations METHODS paragraph 26602 For docking NTD to PIN, OSCAR-star was first used to rebuild sidechains in the head-to-tail PIN dimer. Docking was carried out by surFit (http://sysimm.ifrec.osaka-u.ac.jp/docking/main/) with restraints obtained from NMR data (Fig. 3a,b) as follows. NTD: R56, L58, G59, V86, K87, H88; PIN: V177, F210, T211, R214, F228, I229, V230, K231, L232, F234, D235, S236. Top-scoring model was selected. METHODS paragraph 26996 For docking IL-6 mRNA 3′UTR to the PIN dimer, each domain of the PIN dimer structure was superimposed onto the PIN dimer of the human X-ray structure (PDB ID: 3V34) in order to graft both water molecules and Mg2+ ions to the mouse model. Each IL-6 representative structure was submitted to the HADDOCK 2.0 server, for total of 10 independent jobs. In order to be consistent with the cleavage assay, active residues consisted of all nucleotides in RNA, Mg2+ and W182, R183, K184, R188, R214, R215, K219, R220, and R247 in the protein. Docked models were selected based on the following criteria: one heavy atom within 7, 8, or 9th nucleotide from the 5′ end was <5 Å from the Mg2+ ion on the primary PIN. Further classification was done manually in order to divide the selected models into two clusters. METHODS title_1 27806 Additional Information METHODS paragraph 27829 Accession codes: The crystal structure of the Regnase-1 PIN domain has been deposited in the Protein Data Bank (accession codes: 5H9V (Form I) and 5H9W (Form II)). The chemical shift assignments of the NTD, the ZF domain, and the CTD have been deposited at Biological Magnetic Resonance Bank (accession codes: 25718, 25719, and 25720, respectively), and the coordinates for the ensemble have been deposited in the Protein Data Bank (accession codes: 2N5J, 2N5K, and 2N5L, respectively). METHODS paragraph 28317 How to cite this article: Yokogawa, M. et al. Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions. Sci. Rep. 6, 22324; doi: 10.1038/srep22324 (2016). 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M. 21873640 REF Bioinformatics ref 27 2011 30852 Fast and accurate prediction of protein side-chain conformations SUPPL footnote 30917 Author Contributions F.I. supervised the overall project. M.Y., T.T., Y.E., D.M.S., O.T., S.A. and F.I. designed the research; M.Y. and T.T. performed the research; M.Y., T.T., N.N.N., H.K., K.Y., D.M.S. and F.I. analyzed the data; and M.Y., N.N.N., H.K., K.Y., D.M.S. and F.I. wrote the paper. All authors reviewed the manuscript. srep22324-f1.jpg f1 FIG fig_title_caption 31249 Structural and functional analyses of Regnase-1. 0.99234915 experimental_method cleaner0 2023-07-06T14:15:01Z MESH: Structural and functional analyses 0.9971781 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 srep22324-f1.jpg f1 FIG fig_caption 31298 (a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. Catalytic Asp residues were shown in sticks. (d) Solution structure of the ZF domain. Three Cys residues and one His residue responsible for Zn2+-binding were shown in sticks. (e) Solution structure of the CTD. All the structures were colored in rainbow from N-terminus (blue) to C-terminus (red). (f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA. Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. (g) Binding of Regnase-1 and IL-6 mRNA was plotted. The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f). (h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA. Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1. 0.9957292 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 evidence DUMMY: cleaner0 2023-07-06T13:56:33Z Solution structure 0.99858487 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9967145 evidence cleaner0 2023-07-06T13:56:15Z DUMMY: Crystal structure 0.9985958 structure_element cleaner0 2023-07-06T12:55:51Z SO: PIN 0.7488216 protein_state cleaner0 2023-07-06T14:11:44Z DUMMY: Catalytic 0.98785895 residue_name cleaner0 2023-07-06T14:01:52Z SO: Asp evidence DUMMY: cleaner0 2023-07-06T13:56:33Z Solution structure 0.99852425 structure_element cleaner0 2023-07-06T12:57:35Z SO: ZF 0.9902659 residue_name cleaner0 2023-07-06T14:01:55Z SO: Cys 0.9896445 residue_name cleaner0 2023-07-06T14:01:57Z SO: His evidence DUMMY: cleaner0 2023-07-06T13:56:33Z Solution structure 0.99867463 structure_element cleaner0 2023-07-06T12:57:43Z SO: CTD 0.96567667 evidence cleaner0 2023-07-06T13:56:53Z DUMMY: structures 0.89467937 experimental_method cleaner0 2023-07-06T14:15:05Z MESH: In vitro gel shift binding assay 0.9954107 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:54:16Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:27Z mRNA evidence DUMMY: cleaner0 2023-07-06T13:42:11Z Fluorescence intensity 0.7841645 protein_state cleaner0 2023-07-06T14:11:48Z DUMMY: free 0.9748128 protein_type cleaner0 2023-07-06T12:54:16Z MESH: IL-6 0.99419165 protein_state cleaner0 2023-07-06T13:49:31Z DUMMY: absence of 0.99360347 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.9940892 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:54:17Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:27Z mRNA 0.9351158 protein_type cleaner0 2023-07-06T12:54:17Z MESH: IL-6 evidence DUMMY: cleaner0 2023-07-06T13:42:42Z fluorescence intensities 0.9395353 protein_type cleaner0 2023-07-06T12:54:17Z MESH: IL-6 0.9077164 experimental_method cleaner0 2023-07-06T14:15:08Z MESH: In vitro cleavage assay 0.99444556 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:54:17Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:27Z mRNA evidence DUMMY: cleaner0 2023-07-06T13:42:11Z Fluorescence intensity 0.96524453 protein_state cleaner0 2023-07-06T13:49:26Z DUMMY: uncleaved protein_type MESH: cleaner0 2023-07-06T12:54:17Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:27Z mRNA 0.9879551 protein_state cleaner0 2023-07-06T13:49:30Z DUMMY: absence of 0.9940297 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 srep22324-f2.jpg f2 FIG fig_title_caption 32262 Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1. protein_state DUMMY: cleaner0 2023-07-06T13:11:11Z Head-to-tail oligomeric_state DUMMY: cleaner0 2023-07-06T13:11:19Z oligomer structure_element SO: cleaner0 2023-07-06T12:55:51Z PIN protein_type MESH: cleaner0 2023-07-06T12:55:06Z RNase 0.9969178 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 srep22324-f2.jpg f2 FIG fig_caption 32360 (a) Gel filtration analyses of the PIN domain. Elution volumes of the standard marker proteins were indicated by arrows at the upper part. (b) Dimer structure of the PIN domain. Two PIN molecules in the crystal were colored white and green, respectively. Catalytic residues and mutated residues were shown in sticks. Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed. 0.9257605 experimental_method cleaner0 2023-07-06T14:15:13Z MESH: Gel filtration analyses 0.9976031 structure_element cleaner0 2023-07-06T12:55:51Z SO: PIN 0.9937178 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: Dimer 0.948103 evidence cleaner0 2023-07-06T13:57:00Z DUMMY: structure 0.99757046 structure_element cleaner0 2023-07-06T12:55:51Z SO: PIN 0.52628136 structure_element cleaner0 2023-07-06T12:55:51Z SO: PIN 0.99690825 evidence cleaner0 2023-07-06T13:57:03Z DUMMY: crystal 0.79562724 site cleaner0 2023-07-06T14:04:43Z SO: Catalytic residues 0.99900573 residue_name_number cleaner0 2023-07-06T13:59:22Z DUMMY: R215 protein_type MESH: cleaner0 2023-07-06T12:55:06Z RNase 0.96012145 oligomeric_state cleaner0 2023-07-06T13:54:30Z DUMMY: monomeric 0.5150391 protein_state cleaner0 2023-07-06T13:20:01Z DUMMY: mutants protein_type MESH: cleaner0 2023-07-06T12:54:17Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:27Z mRNA srep22324-f3.jpg f3 FIG fig_title_caption 32894 Domain-domain interaction between the NTD and the PIN domain. 0.99806803 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99878544 structure_element cleaner0 2023-07-06T12:55:51Z SO: PIN srep22324-f3.jpg f3 FIG fig_caption 32956 (a) NMR analyses of the NTD-binding to the PIN domain. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right). Pro and the residues without analysis were colored black and gray, respectively. (b) NMR analyses of the PIN-binding to the NTD. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD. S62 was colored gray and excluded from the analysis, due to low signal intensity. (c) Docking model of the NTD and the PIN domain. The NTD and the PIN domain are shown in cyan and white, respectively. Residues in close proximity (<5 Å) to each other in the docking structure were colored yellow. Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled. experimental_method MESH: cleaner0 2023-07-06T13:57:49Z NMR analyses 0.390678 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD structure_element SO: cleaner0 2023-07-06T12:55:51Z PIN 0.51126844 experimental_method cleaner0 2023-07-06T14:15:18Z MESH: overlaid 0.6816016 evidence cleaner0 2023-07-06T13:57:09Z DUMMY: spectra 0.99837154 structure_element cleaner0 2023-07-06T12:55:51Z SO: PIN 0.9659381 residue_name cleaner0 2023-07-06T14:02:05Z SO: Pro experimental_method MESH: cleaner0 2023-07-06T13:57:50Z NMR analyses structure_element SO: cleaner0 2023-07-06T12:55:51Z PIN 0.9985783 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD evidence DUMMY: cleaner0 2023-07-06T14:05:19Z significant chemical shift changes experimental_method MESH: cleaner0 2023-07-06T13:57:27Z overlaid 0.6049998 evidence cleaner0 2023-07-06T13:57:14Z DUMMY: spectra 0.99854714 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99872404 residue_name_number cleaner0 2023-07-06T13:59:27Z DUMMY: S62 0.99856657 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.9984694 structure_element cleaner0 2023-07-06T12:55:51Z SO: PIN 0.99852055 structure_element cleaner0 2023-07-06T12:55:58Z SO: NTD 0.99842215 structure_element cleaner0 2023-07-06T12:55:51Z SO: PIN 0.87693775 evidence cleaner0 2023-07-06T13:58:03Z DUMMY: docking structure 0.68959475 site cleaner0 2023-07-06T14:04:47Z SO: Catalytic residues structure_element SO: cleaner0 2023-07-06T12:55:51Z PIN evidence DUMMY: cleaner0 2023-07-06T14:05:03Z significant chemical shift changes srep22324-f4.jpg f4 FIG fig_title_caption 33942 Critical residues in the PIN domain for the RNase activity of Regnase-1. structure_element SO: cleaner0 2023-07-06T12:55:51Z PIN protein_type MESH: cleaner0 2023-07-06T12:55:06Z RNase 0.9968206 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 srep22324-f4.jpg f4 FIG fig_caption 34015 (a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA. The results indicate mean ± SD of four independent experiments. (b) In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. Mutated basic residues were shown in sticks and those with significantly reduced RNase activities were colored red or yellow. 0.9900084 experimental_method cleaner0 2023-07-06T14:15:23Z MESH: In vitro cleavage assay protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants protein_type MESH: cleaner0 2023-07-06T12:54:17Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:27Z mRNA 0.9858886 experimental_method cleaner0 2023-07-06T14:15:26Z MESH: In vitro cleavage assay protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants 0.59535795 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 0.65554714 chemical cleaner0 2023-07-06T13:00:27Z CHEBI: mRNA evidence DUMMY: cleaner0 2023-07-06T13:42:11Z fluorescence intensity protein_state DUMMY: cleaner0 2023-07-06T13:49:27Z uncleaved 0.997189 chemical cleaner0 2023-07-06T13:00:27Z CHEBI: mRNA 0.89147466 structure_element cleaner0 2023-07-06T12:55:51Z SO: PIN 0.9949576 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer 0.99110913 evidence cleaner0 2023-07-06T13:58:10Z DUMMY: structure protein_type MESH: cleaner0 2023-07-06T12:55:06Z RNase srep22324-f5.jpg f5 FIG fig_title_caption 34541 Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity. 0.9645019 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_state DUMMY: cleaner0 2023-07-06T13:20:01Z mutants 0.9961874 mutant cleaner0 2023-07-06T13:08:10Z MESH: DDNN 0.6436507 protein_state cleaner0 2023-07-06T13:19:23Z DUMMY: mutant protein_type MESH: cleaner0 2023-07-06T12:55:06Z RNase srep22324-f5.jpg f5 FIG fig_caption 34666 (a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA. (c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN. 0.98775005 experimental_method cleaner0 2023-07-06T14:15:32Z MESH: In vitro cleavage assay 0.99594265 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein_type MESH: cleaner0 2023-07-06T12:54:17Z IL-6 chemical CHEBI: cleaner0 2023-07-06T13:00:27Z mRNA 0.98873675 experimental_method cleaner0 2023-07-06T14:15:35Z MESH: In vitro cleavage assay 0.9957145 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1 protein PR: cleaner0 2023-07-06T12:55:00Z Regnase-1 chemical CHEBI: cleaner0 2023-07-06T13:00:27Z mRNA evidence DUMMY: cleaner0 2023-07-06T13:42:11Z fluorescence intensity protein_state DUMMY: cleaner0 2023-07-06T13:49:27Z uncleaved 0.99734384 chemical cleaner0 2023-07-06T13:00:27Z CHEBI: mRNA 0.9837165 structure_element cleaner0 2023-07-06T12:55:51Z SO: PIN 0.9794327 oligomeric_state cleaner0 2023-07-06T13:11:23Z DUMMY: dimer protein_type MESH: cleaner0 2023-07-06T12:55:06Z RNase protein_state DUMMY: cleaner0 2023-07-06T13:50:30Z presence of mutant MESH: cleaner0 2023-07-06T13:08:10Z DDNN protein_state DUMMY: cleaner0 2023-07-06T13:19:23Z mutant structure_element SO: cleaner0 2023-07-06T12:55:51Z PIN protein_type MESH: cleaner0 2023-07-06T12:55:06Z RNase protein_state DUMMY: cleaner0 2023-07-06T13:50:31Z presence of mutant MESH: cleaner0 2023-07-06T13:08:10Z DDNN protein_state DUMMY: cleaner0 2023-07-06T13:19:23Z mutant structure_element SO: cleaner0 2023-07-06T12:55:51Z PIN srep22324-f6.jpg f6 FIG fig_title_caption 35284 Schematic representation of regulation of the Regnase-1 catalytic activity through the domain-domain interactions. 0.99573964 protein cleaner0 2023-07-06T12:55:00Z PR: Regnase-1