{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T23:27:24Z","timestamp":1790724444203,"version":"4.1.0"},"reference-count":37,"publisher":"Oxford University Press (OUP)","issue":"1","license":[{"start":{"date-parts":[[2022,12,22]],"date-time":"2022-12-22T00:00:00Z","timestamp":1671667200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000054","name":"NCI","doi-asserted-by":"publisher","award":["P01 CA092584"],"award-info":[{"award-number":["P01 CA092584"]}],"id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000289","name":"Cancer Research UK","doi-asserted-by":"publisher","award":["A24759"],"award-info":[{"award-number":["A24759"]}],"id":[{"id":"10.13039\/501100000289","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Universtiy of Oxford"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2023,1,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>NSP14 is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both activities are essential for the viral life cycle and may be targeted for anti-viral therapeutics. NSP14 forms a complex with NSP10, and this interaction enhances the nuclease but not the methyltransferase activity. We have determined the structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 \u00c5 resolution. Comparisons with NSP14\/NSP10 complexes reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. We have determined the structure of NSP14 in complex with cap analogue 7MeGpppG, and observe conformational changes within a SAM\/SAH interacting loop that plays a key role in viral mRNA capping offering new insights into MTase activity. We perform an X-ray fragment screen on NSP14, revealing 72 hits bound to sites of inhibition in the ExoN and MTase domains. These fragments serve as excellent starting point tools for structure guided development of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.<\/jats:p>","DOI":"10.1093\/nar\/gkac1207","type":"journal-article","created":{"date-parts":[[2022,12,12]],"date-time":"2022-12-12T11:37:23Z","timestamp":1670845043000},"page":"475-487","source":"Crossref","is-referenced-by-count":60,"title":["Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development"],"prefix":"10.1093","volume":"51","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5167-3203","authenticated-orcid":false,"given":"Nergis","family":"Imprachim","sequence":"first","affiliation":[{"name":"Centre for Medicines Discovery, University of Oxford , South Parks Rd, Oxford \u00a0OX1 3QU, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuliana","family":"Yosaatmadja","sequence":"additional","affiliation":[{"name":"Centre for Medicines Discovery, University of Oxford , South Parks Rd, Oxford \u00a0OX1 3QU, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4488-0516","authenticated-orcid":false,"given":"Joseph A","family":"Newman","sequence":"additional","affiliation":[{"name":"Centre for Medicines Discovery, University of Oxford , South Parks Rd, Oxford \u00a0OX1 3QU, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2022,12,22]]},"reference":[{"key":"2023011609422045400_B1","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1080\/22221751.2020.1719902","article-title":"Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan","volume":"9","author":"Chan","year":"2020","journal-title":"Emerg. Microbes Infect."},{"key":"2023011609422045400_B2","doi-asserted-by":"crossref","first-page":"e00473-12","DOI":"10.1128\/mBio.00473-12","article-title":"Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans","volume":"3","author":"van\u00a0Boheemen","year":"2012","journal-title":"Mbio"},{"key":"2023011609422045400_B3","doi-asserted-by":"crossref","first-page":"5108","DOI":"10.1073\/pnas.0508200103","article-title":"Discovery of an RNA virus 3\u2032\u21925\u2032 exoribonuclease that is critically involved in coronavirus RNA synthesis","volume":"103","author":"Minskaia","year":"2006","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023011609422045400_B4","doi-asserted-by":"crossref","first-page":"9372","DOI":"10.1073\/pnas.1201130109","article-title":"RNA 3\u2032-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10\/nsp14 exoribonuclease complex","volume":"109","author":"Bouvet","year":"2012","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023011609422045400_B5","doi-asserted-by":"crossref","first-page":"9436","DOI":"10.1073\/pnas.1508686112","article-title":"Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex","volume":"112","author":"Ma","year":"2015","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023011609422045400_B6","doi-asserted-by":"crossref","first-page":"7375","DOI":"10.3390\/ijms21197375","article-title":"Crystal structure of non-structural protein 10 from severe acute respiratory syndrome coronavirus-2","volume":"21","author":"Rogstam","year":"2020","journal-title":"Int. J. Mol. Sci."},{"key":"2023011609422045400_B7","doi-asserted-by":"crossref","first-page":"7902","DOI":"10.1128\/JVI.00483-06","article-title":"Dodecamer structure of severe acute respiratory syndrome coronavirus nonstructural protein nsp10","volume":"80","author":"Su","year":"2006","journal-title":"J. Virol."},{"key":"2023011609422045400_B8","doi-asserted-by":"crossref","first-page":"5382","DOI":"10.1093\/nar\/gkab320","article-title":"Crystal structure of SARS-CoV-2 nsp10 bound to nsp14-ExoN domain reveals an exoribonuclease with both structural and functional integrity","volume":"49","author":"Lin","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"2023011609422045400_B9","doi-asserted-by":"crossref","first-page":"1142","DOI":"10.1126\/science.abi9310","article-title":"Structural basis of mismatch recognition by a SARS-CoV-2 proofreading enzyme","volume":"373","author":"Liu","year":"2021","journal-title":"Science"},{"key":"2023011609422045400_B10","doi-asserted-by":"crossref","first-page":"5619","DOI":"10.1128\/JVI.78.11.5619-5632.2004","article-title":"Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase","volume":"78","author":"Ivanov","year":"2004","journal-title":"J. Virol."},{"key":"2023011609422045400_B11","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1016\/j.cell.2020.11.016","article-title":"Cryo-EM structure of an extended SARS-CoV-2 replication and transcription complex reveals an intermediate state in Cap synthesis","volume":"184","author":"Yan","year":"2020","journal-title":"Cell"},{"key":"2023011609422045400_B12","doi-asserted-by":"crossref","first-page":"3484","DOI":"10.1073\/pnas.0808790106","article-title":"Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase","volume":"106","author":"Chen","year":"2009","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023011609422045400_B13","doi-asserted-by":"crossref","first-page":"e1002294","DOI":"10.1371\/journal.ppat.1002294","article-title":"Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2\u2032-O-methylation by nsp16\/nsp10 protein complex","volume":"7","author":"Chen","year":"2011","journal-title":"PLoS Pathog."},{"key":"2023011609422045400_B14","doi-asserted-by":"crossref","first-page":"3474","DOI":"10.1016\/j.cell.2021.05.033","article-title":"Coupling of N7-methyltransferase and 3\u2032-5\u2032 exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading","volume":"184","author":"Yan","year":"2021","journal-title":"Cell"},{"key":"2023011609422045400_B15","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1107\/S0907444909047337","article-title":"Xds","volume":"66","author":"Kabsch","year":"2010","journal-title":"Acta. Crystallogr. D Biol. Crystallogr."},{"key":"2023011609422045400_B16","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1107\/S0907444912001308","article-title":"Towards automated crystallographic structure refinement with phenix.refine","volume":"68","author":"Afonine","year":"2012","journal-title":"Acta. Crystallogr. D Biol. Crystallogr."},{"key":"2023011609422045400_B17","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1107\/S2059798316020234","article-title":"The XChemExplorer graphical workflow tool for routine or large-scale protein-ligand structure determination","volume":"73","author":"Krojer","year":"2017","journal-title":"Acta Crystallogr D Struct Biol"},{"key":"2023011609422045400_B18","doi-asserted-by":"crossref","first-page":"15123","DOI":"10.1038\/ncomms15123","article-title":"A multi-crystal method for extracting obscured crystallographic states from conventionally uninterpretable electron density","volume":"8","author":"Pearce","year":"2017","journal-title":"Nat. Commun."},{"key":"2023011609422045400_B19","doi-asserted-by":"crossref","first-page":"3717","DOI":"10.1038\/s41467-020-17495-9","article-title":"Structural analysis of the SARS-CoV-2 methyltransferase complex involved in RNA cap creation bound to sinefungin","volume":"11","author":"Krafcikova","year":"2020","journal-title":"Nat. Commun."},{"key":"2023011609422045400_B20","doi-asserted-by":"crossref","first-page":"E162","DOI":"10.1073\/pnas.1718806115","article-title":"Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA","volume":"115","author":"Ferron","year":"2018","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023011609422045400_B21","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1039\/D1CB00135C","article-title":"Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14\/nsp16","volume":"3","author":"Kozielski","year":"2021","journal-title":"RSC Chem. Biol."},{"key":"2023011609422045400_B22","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1016\/S0969-2126(02)00738-4","article-title":"Structural basis for proofreading during replication of the Escherichia coli chromosome","volume":"10","author":"Hamdan","year":"2002","journal-title":"Structure"},{"key":"2023011609422045400_B23","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1002\/j.1460-2075.1991.tb07917.x","article-title":"Structural basis for the 3\u2032-5\u2032 exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism","volume":"10","author":"Beese","year":"1991","journal-title":"EMBO J."},{"key":"2023011609422045400_B24","doi-asserted-by":"crossref","first-page":"e2106379119","DOI":"10.1073\/pnas.2106379119","article-title":"Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN","volume":"119","author":"Moeller","year":"2022","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2023011609422045400_B25","doi-asserted-by":"crossref","first-page":"1484","DOI":"10.1093\/nar\/gkab1303","article-title":"Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification","volume":"50","author":"Baddock","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"2023011609422045400_B26","doi-asserted-by":"crossref","first-page":"1050","DOI":"10.1016\/j.str.2022.04.014","article-title":"Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity","volume":"30","author":"Czarna","year":"2022","journal-title":"Structure"},{"key":"2023011609422045400_B27","doi-asserted-by":"crossref","first-page":"101518","DOI":"10.1016\/j.jbc.2021.101518","article-title":"Activation of the SARS-CoV-2 NSP14 3\u2032-5\u2032 exoribonuclease by NSP10 and response to antiviral inhibitors","volume":"298","author":"Riccio","year":"2022","journal-title":"J. Biol. Chem."},{"key":"2023011609422045400_B28","doi-asserted-by":"crossref","first-page":"1597","DOI":"10.1080\/15476286.2015.1004955","article-title":"RNA methyltransferases involved in 5\u2032 cap biosynthesis","volume":"11","author":"Byszewska","year":"2014","journal-title":"RNA Biol"},{"key":"2023011609422045400_B29","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.virusres.2013.05.001","article-title":"Characterization of the guanine-N7 methyltransferase activity of coronavirus nsp14 on nucleotide GTP","volume":"176","author":"Jin","year":"2013","journal-title":"Virus Res."},{"key":"2023011609422045400_B30","doi-asserted-by":"crossref","first-page":"2322","DOI":"10.1039\/C5SC03115J","article-title":"A poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of PHIP(2), an atypical bromodomain","volume":"7","author":"Cox","year":"2016","journal-title":"Chem. Sci."},{"key":"2023011609422045400_B31","doi-asserted-by":"crossref","DOI":"10.1128\/mBio.00221-18","article-title":"Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease","volume":"9","author":"Agostini","year":"2018","journal-title":"Mbio"},{"key":"2023011609422045400_B32","doi-asserted-by":"crossref","first-page":"1136","DOI":"10.1016\/j.molcel.2020.11.048","article-title":"Coronavirus RNA proofreading: molecular basis and therapeutic targeting","volume":"80","author":"Robson","year":"2020","journal-title":"Mol. Cell"},{"key":"2023011609422045400_B33","doi-asserted-by":"crossref","first-page":"4212","DOI":"10.1021\/acs.jproteome.1c00206","article-title":"Genetic variability of the SARS-CoV-2 pocketome","volume":"20","author":"Yazdani","year":"2021","journal-title":"J. Proteome Res."},{"key":"2023011609422045400_B34","doi-asserted-by":"crossref","first-page":"1200","DOI":"10.1177\/24725552211026261","article-title":"Probing the SAM binding site of SARS-CoV-2 Nsp14 in vitro using SAM competitive inhibitors guides developing selective bisubstrate inhibitors","volume":"26","author":"Devkota","year":"2021","journal-title":"SLAS Discov."},{"key":"2023011609422045400_B35","doi-asserted-by":"crossref","first-page":"2214","DOI":"10.1021\/acsinfecdis.1c00131","article-title":"The structure-based design of SARS-CoV-2 nsp14 methyltransferase ligands yields nanomolar inhibitors","volume":"7","author":"Otava","year":"2021","journal-title":"ACS Infect. Dis."},{"key":"2023011609422045400_B36","doi-asserted-by":"crossref","first-page":"10280","DOI":"10.1128\/JVI.00017-07","article-title":"Processing of open reading frame 1a replicase proteins nsp7 to nsp10 in murine hepatitis virus strain A59 replication","volume":"81","author":"Deming","year":"2007","journal-title":"J. Virol."},{"key":"2023011609422045400_B37","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1016\/j.cell.2020.11.016","article-title":"Cryo-EM structure of an extended SARS-CoV-2 replication and transcription complex reveals an intermediate state in Cap synthesis","volume":"184","author":"Yan","year":"2021","journal-title":"Cell"}],"container-title":["Nucleic Acids Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/51\/1\/475\/48722810\/gkac1207.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/51\/1\/475\/48722810\/gkac1207.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,1,16]],"date-time":"2023-01-16T04:52:27Z","timestamp":1673844747000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/nar\/article\/51\/1\/475\/6956356"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,12,22]]},"references-count":37,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,12,22]]},"published-print":{"date-parts":[[2023,1,11]]}},"URL":"https:\/\/doi.org\/10.1093\/nar\/gkac1207","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2022.03.11.483836","asserted-by":"object"}]},"ISSN":["0305-1048","1362-4962"],"issn-type":[{"value":"0305-1048","type":"print"},{"value":"1362-4962","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2023,1,11]]},"published":{"date-parts":[[2022,12,22]]}}}