{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T19:33:48Z","timestamp":1791920028694,"version":"4.3.4"},"reference-count":38,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2022,6,30]],"date-time":"2022-06-30T00:00:00Z","timestamp":1656547200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001381","name":"National Research Foundation","doi-asserted-by":"publisher","award":["NRF2016NRF-CRP001-063"],"award-info":[{"award-number":["NRF2016NRF-CRP001-063"]}],"id":[{"id":"10.13039\/501100001381","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Nanyang Presidential Graduate Scholarship","award":["NRF2016NRF-CRP001-063"],"award-info":[{"award-number":["NRF2016NRF-CRP001-063"]}]},{"name":"NTU Lee Kong Chian School of Medicine","award":["NRF2016NRF-CRP001-063"],"award-info":[{"award-number":["NRF2016NRF-CRP001-063"]}]},{"name":"A*STAR core funding","award":["NRF2016NRF-CRP001-063"],"award-info":[{"award-number":["NRF2016NRF-CRP001-063"]}]},{"name":"Singapore National Research Foundation","award":["NRF2016NRF-CRP001-063"],"award-info":[{"award-number":["NRF2016NRF-CRP001-063"]}]},{"name":"Career Development Award 2021 (A*STAR BMRC)","award":["NRF2016NRF-CRP001-063"],"award-info":[{"award-number":["NRF2016NRF-CRP001-063"]}]},{"name":"Career Development Fund 2021 (A*STAR BMRC)","award":["NRF2016NRF-CRP001-063"],"award-info":[{"award-number":["NRF2016NRF-CRP001-063"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Diseases caused by flaviviruses such as dengue virus (DENV) and West Nile Virus (WNV), are a serious threat to public health. The flavivirus single-stranded RNA genome is translated into a polyprotein which is cleaved into three structural proteins and seven non-structural proteins by the viral and cellular proteases. Non-structural (NS) protein 3 is a multifunctional protein that has N-terminal protease and C-terminal helicase domains. The NS3 protease requires co-factor NS2B for enzymatic activity and folding. Due to its essential role in viral replication, NS2B-NS3 protease is an attractive target for antiviral drugs. Despite the availability of crystal structures, dynamic interactions of the N- and C-termini of NS2B co-factor have been elusive due to their flexible fold. In this study, we employ integrative structural approaches combined with biochemical assays to elucidate the dynamic interactions of the flexible DENV4 NS2B and NS3 N- and C-termini. We captured the crystal structure of self-cleaved DENV4 NS2B47NS3 protease in post cleavage state. The intermediate conformation adopted in the reported structure can be targeted by allosteric inhibitors. Comparison of our new findings from DENV4 against previously studied ZIKV NS2B-NS3 proteins reveals differences in NS2B-NS3 function between the two viruses. No inhibition of protease activity was observed for unlinked DENV NS2B-NS3 in presence of the cleavage site while ZIKV NS2B-NS3 cleavage inhibits protease activity. Another difference is that binding of the NS2B C-terminus to DENV4 eNS2B47NS3Pro active site is mediated via interactions with P4-P6 residues while for ZIKV, the binding of NS2B C-terminus to active site is mediated by P1-P3 residues. The mapping of NS2B N- and C-termini with NS3 indicates that these intermolecular interactions occur mainly on the beta-barrel 2 of the NS3 protease domain. Our integrative approach enables a comprehensive understanding of the folding and dynamic interactions of DENV NS3 protease and its cofactor NS2B.<\/jats:p>","DOI":"10.3390\/v14071440","type":"journal-article","created":{"date-parts":[[2022,7,1]],"date-time":"2022-07-01T01:40:36Z","timestamp":1656639636000},"page":"1440","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["Dynamic Interactions of Post Cleaved NS2B Cofactor and NS3 Protease Identified by Integrative Structural Approaches"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3827-8580","authenticated-orcid":false,"given":"Jun-Ping","family":"Quek","sequence":"first","affiliation":[{"name":"Lee Kong Chian School of Medicine, Nanyang Technological University, EMB 03-07, 59 Nanyang Drive, Singapore 636921, Singapore"},{"name":"NTU Institute of Structural Biology, Nanyang Technological University, EMB 06-01, 59 Nanyang Drive, Singapore 636921, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4297-7525","authenticated-orcid":false,"given":"Zheng","family":"Ser","sequence":"additional","affiliation":[{"name":"Functional Proteomics Laboratory, SingMass National Laboratory, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 61 Biopolis Drive, #07-03, Singapore 138673, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2748-9366","authenticated-orcid":false,"given":"Bing Liang Alvin","family":"Chew","sequence":"additional","affiliation":[{"name":"Lee Kong Chian School of Medicine, Nanyang Technological University, EMB 03-07, 59 Nanyang Drive, Singapore 636921, Singapore"},{"name":"NTU Institute of Structural Biology, Nanyang Technological University, EMB 06-01, 59 Nanyang Drive, Singapore 636921, Singapore"},{"name":"NTU Institute of Health Technologies, Interdisciplinary Graduate School, Nanyang Technological University, RTP 02-07, 50 Nanyang Drive, Singapore 637553, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Li","sequence":"additional","affiliation":[{"name":"SingMass National Laboratory, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Blk S2, #02, Singapore 117543, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lili","family":"Wang","sequence":"additional","affiliation":[{"name":"SingMass National Laboratory, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Blk S2, #02, Singapore 117543, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Radoslaw M.","family":"Sobota","sequence":"additional","affiliation":[{"name":"Functional Proteomics Laboratory, SingMass National Laboratory, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 61 Biopolis Drive, #07-03, Singapore 138673, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dahai","family":"Luo","sequence":"additional","affiliation":[{"name":"Lee Kong Chian School of Medicine, Nanyang Technological University, EMB 03-07, 59 Nanyang Drive, Singapore 636921, Singapore"},{"name":"NTU Institute of Structural Biology, Nanyang Technological University, EMB 06-01, 59 Nanyang Drive, Singapore 636921, Singapore"},{"name":"School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wint Wint","family":"Phoo","sequence":"additional","affiliation":[{"name":"Functional Proteomics Laboratory, SingMass National Laboratory, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 61 Biopolis Drive, #07-03, Singapore 138673, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1146\/annurev.micro.62.081307.163005","article-title":"Global Spread and Persistence of Dengue","volume":"62","author":"Kyle","year":"2008","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1146\/annurev.mi.44.100190.003245","article-title":"Flavivirus Genome Organization, Expression, and Replication","volume":"44","author":"Chambers","year":"1990","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2467","DOI":"10.1128\/jvi.65.5.2467-2475.1991","article-title":"Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins","volume":"65","author":"Falgout","year":"1991","journal-title":"J. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1535","DOI":"10.1128\/jvi.66.3.1535-1542.1992","article-title":"Cleavage of the dengue virus polyprotein at the NS3\/NS4A and NS4B\/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A\/NS4B may be processed by a cellular protease","volume":"66","author":"Cahour","year":"1992","journal-title":"J. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3794","DOI":"10.1128\/jvi.68.6.3794-3802.1994","article-title":"NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: In vitro and in vivo studies","volume":"68","author":"Amberg","year":"1994","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2206","DOI":"10.1016\/j.febslet.2014.05.018","article-title":"The dengue virus NS2B-NS3 protease retains the closed conformation in the complex with BPTI","volume":"588","author":"Chen","year":"2014","journal-title":"FEBS Lett."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"12895","DOI":"10.1128\/JVI.00942-09","article-title":"Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication","volume":"83","author":"Assenberg","year":"2009","journal-title":"J. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1038\/nsmb1073","article-title":"Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus","volume":"13","author":"Erbel","year":"2006","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1107\/S1744309106001199","article-title":"Purification and crystallization of dengue and West Nile virus NS2B\u2013NS3 complexes. Acta Crystallogr","volume":"62","author":"Chaillet","year":"2006","journal-title":"Sect. F Struct. Biol. Cryst. Commun."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"795","DOI":"10.1110\/ps.072753207","article-title":"Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold","volume":"16","author":"Aleshin","year":"2007","journal-title":"Protein Sci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"19205","DOI":"10.1021\/ja208435s","article-title":"Binding of low molecular weight inhibitors promotes large conformational changes in the dengue virus NS2B-NS3 protease: Fold analysis by pseudocontact shifts","volume":"133","author":"Nguyen","year":"2011","journal-title":"J. Am. Chem. Soc."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Su, X.-C., Ozawa, K., Qi, R., Vasudevan, S., Lim, S.P., and Otting, G. (2009). NMR Analysis of the Dynamic Exchange of the NS2B Cofactor between Open and Closed Conformations of the West Nile Virus NS2B-NS3 Protease. PLoS Negl. Trop. Dis., 3.","DOI":"10.1371\/journal.pntd.0000561"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4244","DOI":"10.1111\/j.1742-4658.2009.07132.x","article-title":"NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease","volume":"276","author":"Su","year":"2009","journal-title":"FEBS J."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"12891","DOI":"10.1074\/jbc.M112.442723","article-title":"NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex","volume":"288","author":"Kim","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"13410","DOI":"10.1038\/ncomms13410","article-title":"Structure of the NS2B-NS3 protease from Zika virus after self-cleavage","volume":"7","author":"Phoo","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"104900","DOI":"10.1016\/j.antiviral.2020.104900","article-title":"Crystal structures of full length DENV4 NS2B-NS3 reveal the dynamic interaction between NS2B and NS3","volume":"182","author":"Phoo","year":"2020","journal-title":"Antivir. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1016\/j.ymeth.2019.03.009","article-title":"Role of integrative structural biology in understanding transcriptional initiation","volume":"159\u2013160","author":"Trnka","year":"2019","journal-title":"Methods"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1146\/annurev.biochem.77.060407.135530","article-title":"Integrating Diverse Data for Structure Determination of Macromolecular Assemblies","volume":"77","author":"Alber","year":"2008","journal-title":"Annu. Rev. Biochem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1380","DOI":"10.1073\/pnas.1120559109","article-title":"Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach","volume":"109","author":"Lasker","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1597","DOI":"10.1126\/science.aai9309","article-title":"Crystal structure of unlinked NS2B-NS3 protease from Zika virus","volume":"354","author":"Zhang","year":"2016","journal-title":"Science"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"116392","DOI":"10.1016\/j.bmc.2021.116392","article-title":"SAR of novel benzothiazoles targeting an allosteric pocket of DENV and ZIKV NS2B\/NS3 proteases","volume":"47","author":"Maus","year":"2021","journal-title":"Bioorganic Med. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1128\/JVI.01788-07","article-title":"Crystal Structure of the NS3 Protease-Helicase from Dengue Virus","volume":"82","author":"Luo","year":"2008","journal-title":"J. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"12883","DOI":"10.1074\/jbc.M611318200","article-title":"Functional Characterization of cis and trans Activity of the Flavivirus NS2B-NS3 Protease","volume":"282","author":"Bera","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"10136","DOI":"10.1073\/pnas.1805195115","article-title":"Targeting intramolecular proteinase NS2B\/3 cleavages for trans-dominant inhibition of dengue virus","volume":"115","author":"Constant","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2524","DOI":"10.1002\/cmdc.202100246","article-title":"Ligand Accessibility Insights to the Dengue Virus NS3-NS2B Protease Assessed by Long-Timescale Molecular Dynamics Simulations","volume":"16","author":"Kronenberger","year":"2021","journal-title":"ChemMedChem"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1204","DOI":"10.1107\/S0907444913000061","article-title":"How good are my data and what is the resolution?","volume":"69","author":"Evans","year":"2013","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1107\/S0907444909052925","article-title":"PHENIX: A comprehensive Python-based system for macromolecular structure solution","volume":"66","author":"Adams","year":"2010","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_28","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":"ref_29","doi-asserted-by":"crossref","first-page":"2126","DOI":"10.1107\/S0907444904019158","article-title":"Coot: Model-building tools for molecular graphics","volume":"60","author":"Emsley","year":"2004","journal-title":"Acta Crystallogr. Sect. D Struct. Biol. Crystallogr."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"15473","DOI":"10.1038\/ncomms15473","article-title":"Optimized fragmentation schemes and data analysis strategies for proteome-wide cross-link identification","volume":"8","author":"Liu","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2545","DOI":"10.1021\/acs.jproteome.9b00085","article-title":"Optimized Cross-Linking Mass Spectrometry for in Situ Interaction Proteomics","volume":"18","author":"Ser","year":"2019","journal-title":"J. Proteome Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2370","DOI":"10.1021\/acs.jproteome.8b00141","article-title":"Identification of MS-Cleavable and Noncleavable Chemically Cross-Linked Peptides with MetaMorpheus","volume":"17","author":"Lu","year":"2018","journal-title":"J. Proteome Res."},{"key":"ref_33","unstructured":"Oliveros, J.C. (2022, April 20). (2007\u20132015). Chap. Available online: https:\/\/bioinfogp.cnb.csic.es\/tools\/venny\/index.html."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/j.jsb.2015.01.014","article-title":"Xlink Analyzer: Software for analysis and visualization of cross-linking data in the context of three-dimensional structures","volume":"189","author":"Kosinski","year":"2015","journal-title":"J. Struct. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera--a visualization system for exploratory research and analysis","volume":"25","author":"Pettersen","year":"2004","journal-title":"J. Comput. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"D1107","DOI":"10.1093\/nar\/gkw1080","article-title":"jPOSTrepo: An international standard data repository for proteomes","volume":"45","author":"Okuda","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1038\/s41592-022-01488-1","article-title":"ColabFold-Making protein folding accessible to all","volume":"19","author":"Mirdita","year":"2022","journal-title":"Nat. Methods"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/7\/1440\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:41:03Z","timestamp":1760139663000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/7\/1440"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,30]]},"references-count":38,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2022,7]]}},"alternative-id":["v14071440"],"URL":"https:\/\/doi.org\/10.3390\/v14071440","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,30]]}}}