{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T08:16:37Z","timestamp":1785226597579,"version":"3.55.0"},"reference-count":47,"publisher":"AIP Publishing","issue":"11","funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["GM098818"],"award-info":[{"award-number":["GM098818"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["pubs.aip.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2020,9,21]]},"abstract":"<jats:p>Broad-spectrum antiviral drugs are urgently needed to stop the Coronavirus Disease 2019 pandemic and prevent future ones. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is related to the SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which have caused the previous outbreaks. The papain-like protease (PLpro) is an attractive drug target due to its essential roles in the viral life cycle. As a cysteine protease, PLpro is rich in cysteines and histidines, and their protonation\/deprotonation modulates catalysis and conformational plasticity. Here, we report the pKa calculations and assessment of the proton-coupled conformational dynamics of SARS-CoV-2 in comparison to SARS-CoV and MERS-CoV PLpros using the recently developed graphical processing unit (GPU)-accelerated implicit-solvent continuous constant pH molecular dynamics method with a new asynchronous replica-exchange scheme, which allows computation on a single GPU card. The calculated pKa\u2019s support the catalytic roles of the Cys\u2013His\u2013Asp triad. We also found that several residues can switch protonation states at physiological pH among which is C270\/271 located on the flexible blocking loop 2 (BL2) of SARS-CoV-2\/CoV PLpro. Simulations revealed that the BL2 can open and close depending on the protonation state of C271\/270, consistent with the most recent crystal structure evidence. Interestingly, despite the lack of an analogous cysteine, BL2 in MERS-CoV PLpro is also very flexible, challenging a current hypothesis. These findings are supported by the all-atom fixed-charge simulations and provide a starting point for more detailed studies to assist the structure-based design of broad-spectrum inhibitors against CoV PLpros.<\/jats:p>","DOI":"10.1063\/5.0020458","type":"journal-article","created":{"date-parts":[[2020,9,15]],"date-time":"2020-09-15T10:46:27Z","timestamp":1600166787000},"update-policy":"https:\/\/doi.org\/10.1063\/aip-crossmark-policy-page","source":"Crossref","is-referenced-by-count":57,"title":["Assessment of proton-coupled conformational dynamics of SARS and MERS\n          coronavirus papain-like proteases: Implication for designing broad-spectrum antiviral\n          inhibitors"],"prefix":"10.1063","volume":"153","author":[{"given":"Jack A.","family":"Henderson","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy , Baltimore, Maryland 21201,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3277-0937","authenticated-orcid":false,"given":"Neha","family":"Verma","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy , Baltimore, Maryland 21201,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1130-6457","authenticated-orcid":false,"given":"Robert C.","family":"Harris","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy , Baltimore, Maryland 21201,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8395-9353","authenticated-orcid":false,"given":"Ruibin","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy , Baltimore, Maryland 21201,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3234-0769","authenticated-orcid":false,"given":"Jana","family":"Shen","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy , Baltimore, Maryland 21201,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"317","published-online":{"date-parts":[[2020,9,15]]},"reference":[{"key":"2025101316470458600_c1","doi-asserted-by":"publisher","first-page":"4562","DOI":"10.1021\/acs.jmedchem.9b01828","article-title":"\u03b1-ketoamides as broad-spectrum inhibitors of\n            coronavirus and enterovirus replication: Structure-based design, synthesis, and activity\n            assessment","volume":"63","year":"2020","journal-title":"J. Med. Chem."},{"key":"2025101316470458600_c2","doi-asserted-by":"publisher","first-page":"4085","DOI":"10.1111\/febs.12936","article-title":"From SARS to MERS:\n            Crystallographic studies on coronaviral proteases enable antiviral drug\n            design","volume":"281","year":"2014","journal-title":"FEBS J."},{"key":"2025101316470458600_c3","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1016\/j.virusres.2007.02.015","article-title":"Evaluating the 3C-like\n            protease activity of SARS-coronavirus: Recommendations for standardized assays for drug\n            discovery","volume":"133","year":"2008","journal-title":"Virus Res."},{"key":"2025101316470458600_c4","doi-asserted-by":"publisher","first-page":"21","DOI":"10.1016\/j.antiviral.2014.12.015","article-title":"The SARS-coronavirus\n            papain-like protease: Structure, function and inhibition by designed antiviral\n            compounds","volume":"115","year":"2015","journal-title":"Antiviral Res."},{"key":"2025101316470458600_c5","doi-asserted-by":"publisher","first-page":"523","DOI":"10.1038\/nrmicro.2016.81","article-title":"SARS and MERS: Recent insights\n            into emerging coronaviruses","volume":"14","year":"2016","journal-title":"Nat. Rev. Microbiol."},{"key":"2025101316470458600_c6","doi-asserted-by":"publisher","first-page":"58","DOI":"10.1016\/j.antiviral.2017.11.001","article-title":"Nsp3 of coronaviruses:\n            Structures and functions of a large multi-domain protein","volume":"149","year":"2018","journal-title":"Antiviral Res."},{"key":"2025101316470458600_c7","doi-asserted-by":"publisher","first-page":"64","DOI":"10.1016\/j.virol.2013.11.040","article-title":"MERS-CoV papain-like protease\n            has deISGylating and deubiquitinating activities","volume":"450-451","year":"2014","journal-title":"Virology"},{"key":"2025101316470458600_c8","doi-asserted-by":"publisher","first-page":"3190","DOI":"10.1002\/1873-3468.12827","article-title":"RNA-virus proteases\n            counteracting host innate immunity","volume":"591","year":"2017","journal-title":"FEBS Lett."},{"key":"2025101316470458600_c9","doi-asserted-by":"publisher","first-page":"4619","DOI":"10.1128\/jvi.02406-09","article-title":"Deubiquitinating and interferon\n            antagonism activities of coronavirus papain-like proteases","volume":"84","year":"2010","journal-title":"J.\n            Virol."},{"key":"2025101316470458600_c10","doi-asserted-by":"publisher","first-page":"32208","DOI":"10.1074\/jbc.m704870200","article-title":"Regulation of IRF-3-dependent\n            innate immunity by the papain-like protease domain of the severe acute respiratory\n            syndrome coronavirus","volume":"282","year":"2007","journal-title":"J. Biol. Chem."},{"key":"2025101316470458600_c11","doi-asserted-by":"publisher","first-page":"10349","DOI":"10.1021\/bi0504761","article-title":"Papain-like protease 2 (PLP2)\n            from severe acute respiratory syndrome coronavirus (SARS-CoV): Expression, purification,\n            characterization, and inhibition","volume":"44","year":"2005","journal-title":"Biochemistry"},{"key":"2025101316470458600_c12","doi-asserted-by":"publisher","first-page":"15189","DOI":"10.1128\/jvi.79.24.15189-15198.2005","article-title":"The papain-like protease of\n            severe acute respiratory syndrome coronavirus has deubiquitinating\n            activity","volume":"79","year":"2005","journal-title":"J. Virol."},{"key":"2025101316470458600_c13","doi-asserted-by":"publisher","first-page":"5717","DOI":"10.1073\/pnas.0510851103","article-title":"Severe acute respiratory\n            syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating\n            enzyme","volume":"103","year":"2006","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2025101316470458600_c14","doi-asserted-by":"publisher","first-page":"237","DOI":"10.1146\/annurev-virology-110615-042301","article-title":"Structure, function, and evolution\n            of coronavirus spike proteins","volume":"3","year":"2016","journal-title":"Annu. Rev. Virol."},{"key":"2025101316470458600_c15","doi-asserted-by":"publisher","first-page":"271.e8","DOI":"10.1016\/j.cell.2020.02.052","article-title":"SARS-CoV-2 cell entry depends\n            on ACE2 and TMPRSS2 and is blocked by a clinically proven protease\n            inhibitor","volume":"181","year":"2020","journal-title":"Cell"},{"key":"2025101316470458600_c16","doi-asserted-by":"publisher","first-page":"2617","DOI":"10.1021\/ct200146j","article-title":"Continuous constant pH molecular\n            dynamics in explicit solvent with pH-based replica exchange","volume":"7","year":"2011","journal-title":"J.\n            Chem. Theory Comput."},{"key":"2025101316470458600_c17","doi-asserted-by":"publisher","first-page":"9543","DOI":"10.1021\/jacs.5b05891","article-title":"pH-dependent population shift\n            regulates BACE1 activity and inhibition","volume":"137","year":"2015","journal-title":"J. Am. Chem.\n            Soc."},{"key":"2025101316470458600_c18","doi-asserted-by":"publisher","first-page":"944","DOI":"10.1021\/acs.jpclett.6b00137","article-title":"Constant pH molecular dynamics\n            reveals pH-modulated binding of two small-molecule BACE1 inhibitors","volume":"7","year":"2016","journal-title":"J. Phys. Chem. Lett."},{"key":"2025101316470458600_c19","doi-asserted-by":"publisher","first-page":"4832","DOI":"10.1021\/acs.jpclett.7b02309","article-title":"Proton-coupled conformational\n            allostery modulates the inhibitor selectivity for\n            \u03b2-secretase","volume":"8","year":"2017","journal-title":"J. Phys. Chem. Lett."},{"key":"2025101316470458600_c20","doi-asserted-by":"publisher","first-page":"5440","DOI":"10.1021\/acs.jpclett.8b02440","article-title":"How ligand protonation state\n            controls water in protein-ligand binding","volume":"9","year":"2018","journal-title":"J. Phys. Chem.\n            Lett."},{"key":"2025101316470458600_c21","doi-asserted-by":"publisher","first-page":"1372","DOI":"10.1021\/acs.jcim.8b00227","article-title":"Generalized Born based continuous\n            constant pH molecular dynamics in amber: Implementation, benchmarking and\n            analysis","volume":"58","year":"2018","journal-title":"J. Chem. Inf. Model."},{"key":"2025101316470458600_c22","year":"2018"},{"key":"2025101316470458600_c23","doi-asserted-by":"publisher","first-page":"1456","DOI":"10.1021\/cb500917m","article-title":"Inhibitor recognition\n            specificity of MERS-CoV papain-like protease may differ from that of\n            SARS-CoV","volume":"10","year":"2015","journal-title":"ACS Chem. Biol."},{"key":"2025101316470458600_c24","doi-asserted-by":"publisher","first-page":"1545","DOI":"10.1002\/jcc.21287","article-title":"CHARMM: The biomolecular\n            simulation program","volume":"30","year":"2009","journal-title":"J. Comput. 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Soc."},{"key":"2025101316470458600_c32","doi-asserted-by":"publisher","first-page":"738","DOI":"10.1002\/prot.20128","article-title":"Constant-pH molecular dynamics using\n            continuous titration coordinates","volume":"56","year":"2004","journal-title":"Proteins"},{"key":"2025101316470458600_c33","doi-asserted-by":"publisher","first-page":"141","DOI":"10.1529\/biophysj.105.061341","article-title":"Constant pH\n            molecular dynamics with proton tautomerism","volume":"89","year":"2005","journal-title":"Biophys.\n            J."},{"key":"2025101316470458600_c34","doi-asserted-by":"publisher","first-page":"9363","DOI":"10.1021\/bi060706r","article-title":"Toward the accurate first-principles\n            prediction of ionization equilibria in proteins","volume":"45","year":"2006","journal-title":"Biochemistry"},{"key":"2025101316470458600_c35","doi-asserted-by":"publisher","first-page":"236","DOI":"10.1016\/j.cpc.2015.06.010","article-title":"Asynchronous replica exchange\n            software for grid and heterogeneous computing","volume":"196","year":"2015","journal-title":"Comput. Phys.\n            Commun."},{"key":"2025101316470458600_c36","doi-asserted-by":"publisher","first-page":"3689","DOI":"10.1021\/acs.jctc.0c00258","article-title":"Predicting reactive cysteines\n            with implicit-solvent-based continuous constant pH molecular dynamics in\n            amber","volume":"16","year":"2020","journal-title":"J. Chem. Theory Comput."},{"key":"2025101316470458600_c37","doi-asserted-by":"publisher","first-page":"926","DOI":"10.1063\/1.445869","article-title":"Comparison of simple potential\n            functions for simulating liquid water","volume":"79","year":"1983","journal-title":"J. Chem. Phys."},{"key":"2025101316470458600_c38","doi-asserted-by":"publisher","first-page":"10089","DOI":"10.1063\/1.464397","article-title":"Particle mesh Ewald: An\n              N log(N) method for Ewald sums in large\n            systems","volume":"98","year":"1993","journal-title":"J. Chem. Phys."},{"key":"2025101316470458600_c39","doi-asserted-by":"publisher","first-page":"3684","DOI":"10.1063\/1.448118","article-title":"Molecular dynamics with coupling\n            to an external bath","volume":"81","year":"1984","journal-title":"J. Chem. Phys."},{"key":"2025101316470458600_c40","doi-asserted-by":"publisher","first-page":"3084","DOI":"10.1021\/ct400341p","article-title":"PTRAJ and CPPTRAJ: Software\n            for processing and analysis of molecular dynamics trajectory data","volume":"9","year":"2013","journal-title":"J. Chem. Theory Comput."},{"key":"2025101316470458600_c41","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1016\/j.antiviral.2014.06.011","article-title":"Crystal structure of the\n            papain-like protease of MERS coronavirus reveals unusual, potentially druggable\n            active-site features","volume":"109","year":"2014","journal-title":"Antiviral Res."},{"key":"2025101316470458600_c42","doi-asserted-by":"publisher","first-page":"614","DOI":"10.1093\/bioinformatics\/btv607","article-title":"DelPhiPKa web server:\n            Predicting pKa of proteins, RNAs and DNAs","volume":"32","year":"2016","journal-title":"Bioinformatics"},{"key":"2025101316470458600_c43","doi-asserted-by":"publisher","first-page":"2186","DOI":"10.1002\/prot.24935","article-title":"pKa\n            predictions for proteins, RNAs, and DNAs with the Gaussian dielectric function using\n            DelPhi pKa","volume":"83","year":"2015","journal-title":"Proteins"},{"key":"2025101316470458600_c44","doi-asserted-by":"publisher","first-page":"1277","DOI":"10.1002\/prot.25608","article-title":"DelPhiPKa: Including salt in\n            the calculations and enabling polar residues to titrate","volume":"86","year":"2018","journal-title":"Proteins"},{"key":"2025101316470458600_c45","doi-asserted-by":"publisher","first-page":"3260","DOI":"10.1002\/prot.23189","article-title":"Progress in the prediction of\n              pKa values in proteins","volume":"79","year":"2011","journal-title":"Proteins"},{"key":"2025101316470458600_c46","doi-asserted-by":"publisher","first-page":"2393","DOI":"10.1021\/jm401712t","article-title":"X-ray structural and\n            biological evaluation of a series of potent and highly selective inhibitors of human\n            coronavirus papain-like proteases","volume":"57","year":"2014","journal-title":"J. Med. Chem."},{"key":"2025101316470458600_c47","doi-asserted-by":"publisher","first-page":"5411","DOI":"10.1021\/acs.jctc.6b00552","article-title":"All-atom continuous constant pH\n            molecular dynamics with particle mesh Ewald and titratable water","volume":"12","year":"2016","journal-title":"J. Chem. Theory Comput."}],"container-title":["The Journal of Chemical Physics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.aip.org\/aip\/jcp\/article-pdf\/doi\/10.1063\/5.0020458\/20749457\/115101_1_5.0020458.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.aip.org\/aip\/jcp\/article-pdf\/doi\/10.1063\/5.0020458\/20749457\/115101_1_5.0020458.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T20:47:11Z","timestamp":1760388431000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.aip.org\/jcp\/article\/153\/11\/115101\/199630\/Assessment-of-proton-coupled-conformational"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,9,15]]},"references-count":47,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2020,9,21]]}},"URL":"https:\/\/doi.org\/10.1063\/5.0020458","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2020.06.30.181305","asserted-by":"object"}]},"ISSN":["0021-9606","1089-7690"],"issn-type":[{"value":"0021-9606","type":"print"},{"value":"1089-7690","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2020,9,21]]},"published":{"date-parts":[[2020,9,15]]},"article-number":"115101"}}