{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:13:19Z","timestamp":1785409999399,"version":"3.56.0"},"posted":{"date-parts":[[2026,5,20]]},"group-title":"Biophysics","reference-count":51,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T00:00:00Z","timestamp":1779235200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["R01AI166050"],"award-info":[{"award-number":["R01AI166050"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>SARS-CoV-2 nonstructural protein 13 (nsp13) is a highly conserved helicase that couples ATP hydrolysis to RNA translocation through long-range allosteric communication between its ATPase and RNA-binding domains. In prior work, we identified L405 as a key regulator of interdomain motions and proposed that the L405D mutation would disrupt this coupling by perturbing conformational translocations required for translocation [J. Phys. Chem. B 2024 v128 492\u2013503]. Subsequent experiments confirmed that L405D attenuates helicase activity while largely preserving ATPase activity, implicating a breakdown in ATP-to-RNA coupling [J. Biol. Chem. 2026 v302 111198]. Here, we provide a data-driven explanation for this decoupling by combining Gaussian accelerated molecular dynamics (GaMD) simulations with Shape-GMM clustering and linear discriminant analysis. Whereas wild-type nsp13 exhibits both conformational selection and induction, L405D collapses the conformational landscape to operate predominantly through selection, eliminating ATP-induced structural transitions required for efficient catalytic cycling. This loss of induction traps the ATP-binding pocket in a mid-open conformation, impairing product release and reducing ATP turnover, while simultaneously disrupting coordinated motif\u2013RNA interactions required for inchworm translocation. These findings establish that mutation-induced reshaping of conformational ensembles can modulate access to reaction-competent states, providing a general framework for understanding how targeted mutations disrupt catalytic function through allosteric ensemble remodeling in motor proteins.<\/jats:p>","DOI":"10.64898\/2026.05.18.726016","type":"posted-content","created":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T04:30:14Z","timestamp":1779337814000},"source":"Crossref","is-referenced-by-count":0,"title":["Mechanochemical Decoupling of ATP Hydrolysis and RNA Translocation in SARS-CoV-2 nsp13 by the L405D Mutation"],"prefix":"10.64898","author":[{"given":"Elham","family":"Fazelpour","sequence":"first","affiliation":[{"name":"Department of Chemistry, Oklahoma State University, Stillwater, OK 74078, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2298-4883","authenticated-orcid":false,"given":"Priti","family":"Roy","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Oklahoma State University, Stillwater, OK 74078, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kole J.","family":"Frederick","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Oklahoma State University, Stillwater, OK 74078, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8603-4388","authenticated-orcid":false,"given":"Martin","family":"McCullagh","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Oklahoma State University, Stillwater, OK 74078, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026060409500914000_2026.05.18.726016v2.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.65.070196.001125"},{"key":"2026060409500914000_2026.05.18.726016v2.2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.76.052305.115300"},{"key":"2026060409500914000_2026.05.18.726016v2.3","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.78.11.5619-5632.2004"},{"key":"2026060409500914000_2026.05.18.726016v2.4","doi-asserted-by":"publisher","DOI":"10.1016\/J.CELL.2020.07.033"},{"key":"2026060409500914000_2026.05.18.726016v2.5","doi-asserted-by":"publisher","DOI":"10.1016\/bs.enz.2021.09.005"},{"key":"2026060409500914000_2026.05.18.726016v2.6","doi-asserted-by":"crossref","first-page":"9144","DOI":"10.1021\/acs.jpclett.0c02421","article-title":"Discovery of COVID-19 inhibitors targeting the SARS-CoV-2 Nsp13 helicase","volume":"11","year":"2020","journal-title":"The Journal of Physical Chemistry Letters"},{"key":"2026060409500914000_2026.05.18.726016v2.7","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.abj4526"},{"key":"2026060409500914000_2026.05.18.726016v2.8","doi-asserted-by":"publisher","DOI":"10.1128\/JB.184.7.1819-1826.2002"},{"key":"2026060409500914000_2026.05.18.726016v2.9","doi-asserted-by":"publisher","DOI":"10.1016\/0968-0004(92)90337-9"},{"key":"2026060409500914000_2026.05.18.726016v2.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2010.03.011"},{"key":"2026060409500914000_2026.05.18.726016v2.11","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2014.12.001"},{"key":"2026060409500914000_2026.05.18.726016v2.12","doi-asserted-by":"crossref","unstructured":"Walker, P. 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C. ; others Changes to virus taxonomy and the Statutes ratified by the International Committee on Taxonomy of Viruses (2020). 2020.","DOI":"10.1007\/s00705-020-04752-x"},{"key":"2026060409500914000_2026.05.18.726016v2.13","doi-asserted-by":"crossref","first-page":"4848","DOI":"10.1038\/s41467-021-25166-6","article-title":"; others Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase","volume":"12","year":"2021","journal-title":"Nature Communications"},{"key":"2026060409500914000_2026.05.18.726016v2.14","doi-asserted-by":"crossref","first-page":"8787","DOI":"10.1021\/acs.jpcb.1c04528","article-title":"Role of ATP in the RNA translocation mechanism of SARS-CoV-2 NSP13 helicase","volume":"125","year":"2021","journal-title":"The Journal of Physical Chemistry B"},{"key":"2026060409500914000_2026.05.18.726016v2.15","first-page":"1","article-title":"Allosteric Regulation of RNA Affinity by Motif V-VI Coupling in West Nile Virus NS3 Helicase","volume":"0","year":"2026","journal-title":"Proteins: Structure, Function, and Bioinformatics"},{"key":"2026060409500914000_2026.05.18.726016v2.16","doi-asserted-by":"crossref","first-page":"492","DOI":"10.1021\/acs.jpcb.3c06714","article-title":"Role of ATP hydrolysis and product release in the translocation mechanism of SARS-CoV-2 NSP13","volume":"128","year":"2024","journal-title":"The Journal of Physical Chemistry B"},{"key":"2026060409500914000_2026.05.18.726016v2.17","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.5b00562"},{"key":"2026060409500914000_2026.05.18.726016v2.18","doi-asserted-by":"publisher","DOI":"10.1002\/anie.199423751"},{"key":"2026060409500914000_2026.05.18.726016v2.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpc.2017.01.004"},{"key":"2026060409500914000_2026.05.18.726016v2.20","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1038\/nchembio.232","article-title":"The role of dynamic conformational ensembles in biomolecular recognition","volume":"5","year":"2009","journal-title":"Nature Chemical Biology"},{"key":"2026060409500914000_2026.05.18.726016v2.21","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2010.04.009"},{"key":"2026060409500914000_2026.05.18.726016v2.22","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(65)80285-6"},{"key":"2026060409500914000_2026.05.18.726016v2.23","doi-asserted-by":"publisher","DOI":"10.1038\/nature04331"},{"key":"2026060409500914000_2026.05.18.726016v2.24","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2010.11.034"},{"key":"2026060409500914000_2026.05.18.726016v2.25","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80716-3"},{"key":"2026060409500914000_2026.05.18.726016v2.26","doi-asserted-by":"publisher","DOI":"10.1016\/S0968-0004(00)01734-5"},{"key":"2026060409500914000_2026.05.18.726016v2.27","doi-asserted-by":"crossref","unstructured":"Mingroni, M. 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