Insights into the mechanism of SARS-CoV-2 main protease autocatalytic maturation from model precursors

Aniana, Annie; Nashed, Nashaat T.; Ghirlando, Rodolfo; Coates, Leighton; Kneller, Daniel W.; Kovalevsky, Andrey; Louis, John M.

doi:10.1038/s42003-023-05469-8

Title: Insights into the mechanism of SARS-CoV-2 main protease autocatalytic maturation from model precursors

Journal Article · 12 November 2023 · Communications Biology

DOI: https://doi.org/10.1038/s42003-023-05469-8 · OSTI ID:2217736

Aniana, Annie ^[1];

^[1];

^[2]; Kneller, Daniel W. ^[3];

^[2];

^[1]

National Institutes of Health (NIH), Bethesda, MD (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
New England Biolabs, Ipswich, MA (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

A critical step for SARS-CoV-2 assembly and maturation involves the autoactivation of the main protease (MPro^WT) from precursor polyproteins. Upon expression, a model precursor of MPro^WT mediates its own release at its termini rapidly to yield a mature dimer. A construct with an E290A mutation within MPro exhibits time dependent autoprocessing of the accumulated precursor at the N-terminal nsp4/nsp5 site followed by the C-terminal nsp5/nsp6 cleavage. In contrast, a precursor containing E290A and R298A mutations (MPro^M) displays cleavage only at the nsp4/nsp5 site to yield an intermediate monomeric product, which is cleaved at the nsp5/nsp6 site only by MPro^WT. MPro^M and the catalytic domain (MPro^1-199) fused to the truncated nsp4 region also show time-dependent conversion in vitro to produce MPro^M and MPro^1-199, respectively. The reactions follow first-order kinetics indicating that the nsp4/nsp5 cleavage occurs via an intramolecular mechanism. These results support a mechanism involving an N-terminal intramolecular cleavage leading to an increase in the dimer population and followed by an intermolecular cleavage at the C-terminus. Thus, targeting the predominantly monomeric MPro precursor for inhibition may lead to the identification of potent drugs for treatment.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Spallation Neutron Source (SNS); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). High Flux Isotope Reactor (HFIR); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Center for Structural Molecular Biology (CSMB)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 2217736

Journal Information:: Communications Biology, Journal Name: Communications Biology Journal Issue: 1 Vol. 6; ISSN 2399-3642

Publisher:: Springer NatureCopyright Statement

Country of Publication:: United States

Language:: English

References (45)

Localization and Membrane Topology of Coronavirus Nonstructural Protein 4: Involvement of the Early Secretory Pathway in Replication Oostra, M.; te Lintelo, E. G.; Deijs, M. Journal of Virology, Vol. 81, Issue 22 https://doi.org/10.1128/JVI.01506-07	journal	November 2007
Structural plasticity of SARS-CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography Kneller, Daniel W.; Phillips, Gwyndalyn; O’Neill, Hugh M. Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-16954-7	journal	June 2020
The establishment of reference sequence for SARS‐CoV‐2 and variation analysis Wang, Changtai; Liu, Zhongping; Chen, Zixiang Journal of Medical Virology, Vol. 92, Issue 6 https://doi.org/10.1002/jmv.25762	journal	March 2020
Correlation between dissociation and catalysis of SARS-CoV main protease Lin, Pei-Ying; Chou, Chi-Yuan; Chang, Hui-Chuan Archives of Biochemistry and Biophysics, Vol. 472, Issue 1 https://doi.org/10.1016/j.abb.2008.01.023	journal	April 2008
Production of Authentic SARS-CoV Mpro with Enhanced Activity: Application as a Novel Tag-cleavage Endopeptidase for Protein Overproduction Xue, Xiaoyu; Yang, Haitao; Shen, Wei Journal of Molecular Biology, Vol. 366, Issue 3 https://doi.org/10.1016/j.jmb.2006.11.073	journal	February 2007
Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag-Pol polyprotein. Louis, J. M.; Nashed, N. T.; Parris, K. D. Proceedings of the National Academy of Sciences, Vol. 91, Issue 17 https://doi.org/10.1073/pnas.91.17.7970	journal	August 1994
A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process Noske, G. D.; Nakamura, A. M.; Gawriljuk, V. O. Journal of Molecular Biology, Vol. 433, Issue 18 https://doi.org/10.1016/j.jmb.2021.167118	journal	September 2021
Crystallographic structure of wild-type SARS-CoV-2 main protease acyl-enzyme intermediate with physiological C-terminal autoprocessing site Lee, Jaeyong; Worrall, Liam J.; Vuckovic, Marija Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-19662-4	journal	November 2020
The Proteins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19 Yoshimoto, Francis K. The Protein Journal, Vol. 39, Issue 3 https://doi.org/10.1007/s10930-020-09901-4	journal	May 2020
MolProbity : all-atom structure validation for macromolecular crystallography Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1 https://doi.org/10.1107/S0907444909042073	journal	December 2009
Phaser crystallographic software McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Journal of Applied Crystallography, Vol. 40, Issue 4 https://doi.org/10.1107/S0021889807021206	journal	July 2007
Current developments in Coot for macromolecular model building of Electron Cryo‐microscopy and Crystallographic Data Casañal, Ana; Lohkamp, Bernhard; Emsley, Paul Protein Science, Vol. 29, Issue 4 https://doi.org/10.1002/pro.3791	journal	March 2020
Autoprocessing and oxyanion loop reorganization upon GC373 and nirmatrelvir binding of monomeric SARS-CoV-2 main protease catalytic domain Nashed, Nashaat T.; Kneller, Daniel W.; Coates, Leighton Communications Biology, Vol. 5, Issue 1 https://doi.org/10.1038/s42003-022-03910-y	journal	September 2022
HIV-I protease: Maturation, enzyme specificity, and drug resistance Louis, John M.; Webert, Irene T.; Tözsér, József HIV-1: Molecular Biology and Pathogenesis https://doi.org/10.1016/S1054-3589(00)49025-3	book	January 2000
Activation and maturation of SARS-CoV main protease Xia, Bin; Kang, Xue Protein & Cell, Vol. 2, Issue 4 https://doi.org/10.1007/s13238-011-1034-1	journal	April 2011
Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix Liebschner, Dorothee; Afonine, Pavel V.; Baker, Matthew L. Acta Crystallographica Section D Structural Biology, Vol. 75, Issue 10 https://doi.org/10.1107/S2059798319011471	journal	October 2019
Overview of the CCP 4 suite and current developments Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D. Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444910045749	journal	March 2011
Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors Zhang, Linlin; Lin, Daizong; Sun, Xinyuanyuan Science https://doi.org/10.1126/science.abb3405	journal	March 2020
Mechanism of the Maturation Process of SARS-CoV 3CL Protease Hsu, Min-Feng; Kuo, Chih-Jung; Chang, Kai-Ti Journal of Biological Chemistry, Vol. 280, Issue 35 https://doi.org/10.1074/jbc.M502577200	journal	March 2005
Mechanism for Controlling the Dimer-Monomer Switch and Coupling Dimerization to Catalysis of the Severe Acute Respiratory Syndrome Coronavirus 3C-Like Protease Shi, J.; Sivaraman, J.; Song, J. Journal of Virology, Vol. 82, Issue 9 https://doi.org/10.1128/JVI.02680-07	journal	February 2008
Topology and Membrane Anchoring of the Coronavirus Replication Complex: Not All Hydrophobic Domains of nsp3 and nsp6 Are Membrane Spanning Oostra, Monique; Hagemeijer, Marne C.; van Gent, Michiel Journal of Virology, Vol. 82, Issue 24 https://doi.org/10.1128/JVI.01219-08	journal	December 2008
Evidence for intramolecular self-cleavage of picornaviral replicase precursors Palmenberg, A. C.; Rueckert, R. R. Journal of Virology, Vol. 41, Issue 1 https://doi.org/10.1128/jvi.41.1.244-249.1982	journal	January 1982
HIV‐1 Protease: Structure, Dynamics, and Inhibition Louis, John M.; Ishima, Rieko; Torchia, Dennis A. Advances in Pharmacology https://doi.org/10.1016/S1054-3589(07)55008-8	book	June 2007
Michaelis-like complex of SARS-CoV-2 main protease visualized by room-temperature X-ray crystallography Kneller, Daniel W.; Zhang, Qiu; Coates, Leighton IUCrJ, Vol. 8, Issue 6 https://doi.org/10.1107/S2052252521010113	journal	October 2021
SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity Muramatsu, Tomonari; Takemoto, Chie; Kim, Yong-Tae Proceedings of the National Academy of Sciences, Vol. 113, Issue 46 https://doi.org/10.1073/pnas.1601327113	journal	October 2016
A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G Gronenborn, A.; Filpula, D.; Essig, N. Science, Vol. 253, Issue 5020 https://doi.org/10.1126/science.1871600	journal	August 1991
Intracellular andin Vitro-Translated 27-kDa Proteins Contain the 3C-like Proteinase Activity of the Coronavirus MHV-A59 Lu, Xiaotao; Lu, Yiqi; Denison, Mark R. Virology, Vol. 222, Issue 2 https://doi.org/10.1006/viro.1996.0434	journal	August 1996
Targeting the Dimerization of the Main Protease of Coronaviruses: A Potential Broad-Spectrum Therapeutic Strategy Goyal, Bhupesh; Goyal, Deepti ACS Combinatorial Science, Vol. 22, Issue 6 https://doi.org/10.1021/acscombsci.0c00058	journal	May 2020
Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor Nashed, Nashaat T.; Aniana, Annie; Ghirlando, Rodolfo Communications Biology, Vol. 5, Issue 1 https://doi.org/10.1038/s42003-022-03084-7	journal	March 2022
Covalent narlaprevir- and boceprevir-derived hybrid inhibitors of SARS-CoV-2 main protease Kneller, Daniel W.; Li, Hui; Phillips, Gwyndalyn Nature Communications, Vol. 13, Issue 1 https://doi.org/10.1038/s41467-022-29915-z	journal	April 2022
Structural, Electronic, and Electrostatic Determinants for Inhibitor Binding to Subsites S1 and S2 in SARS-CoV-2 Main Protease Kneller, Daniel W.; Li, Hui; Galanie, Stephanie Journal of Medicinal Chemistry, Vol. 64, Issue 23 https://doi.org/10.1021/acs.jmedchem.1c01475	journal	October 2021
How good are my data and what is the resolution? Evans, Philip R.; Murshudov, Garib N. Acta Crystallographica Section D Biological Crystallography, Vol. 69, Issue 7 https://doi.org/10.1107/S0907444913000061	journal	June 2013
Molecular mechanisms of severe acute respiratory syndrome (SARS) Groneberg, David A.; Hilgenfeld, Rolf; Zabel, Peter Respiratory Research, Vol. 6, Issue 1 https://doi.org/10.1186/1465-9921-6-8	journal	January 2005
Mutation of Glu-166 Blocks the Substrate-Induced Dimerization of SARS Coronavirus Main Protease Cheng, Shu-Chun; Chang, Gu-Gang; Chou, Chi-Yuan Biophysical Journal, Vol. 98, Issue 7 https://doi.org/10.1016/j.bpj.2009.12.4272	journal	April 2010
Liberation of SARS-CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode Chen, Shuai; Jonas, Felix; Shen, Can Protein & Cell, Vol. 1, Issue 1 https://doi.org/10.1007/s13238-010-0011-4	journal	January 2010
Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor Kovalevsky, Andrey; Coates, Leighton; Kneller, Daniel W. Journal of Molecular Biology, Vol. 434, Issue 24 https://doi.org/10.1016/j.jmb.2022.167876	journal	December 2022
electronic Ligand Builder and Optimization Workbench ( eLBOW ): a tool for ligand coordinate and restraint generation Moriarty, Nigel W.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Acta Crystallographica Section D Biological Crystallography, Vol. 65, Issue 10 https://doi.org/10.1107/S0907444909029436	journal	September 2009
Self-cleaving proteases Blair, Wade S.; Semler, Bert L. Current Opinion in Cell Biology, Vol. 3, Issue 6 https://doi.org/10.1016/0955-0674(91)90126-J	journal	December 1991
Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography Kneller, Daniel W.; Phillips, Gwyndalyn; Weiss, Kevin L. Journal of Biological Chemistry, Vol. 295, Issue 50 https://doi.org/10.1074/jbc.AC120.016154	journal	October 2020
Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease Ma, Chunlong; Sacco, Michael Dominic; Hurst, Brett Cell Research, Vol. 30, Issue 8 https://doi.org/10.1038/s41422-020-0356-z	journal	June 2020
Recognition of Divergent Viral Substrates by the SARS-CoV-2 Main Protease MacDonald, Elizabeth A.; Frey, Gary; Namchuk, Mark N. ACS Infectious Diseases, Vol. 7, Issue 9 https://doi.org/10.1021/acsinfecdis.1c00237	journal	August 2021
The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle Ricciardi, Simona; Guarino, Andrea Maria; Giaquinto, Laura Nature, Vol. 606, Issue 7915 https://doi.org/10.1038/s41586-022-04835-6	journal	May 2022
SARS-CoV-2 M ^pro inhibitors with antiviral activity in a transgenic mouse model Qiao, Jingxin; Li, Yue-Shan; Zeng, Rui Science, Vol. 371, Issue 6536 https://doi.org/10.1126/science.abf1611	journal	February 2021
Structural Characterization of SARS-CoV-2: Where We Are, and Where We Need to Be Mariano, Giuseppina; Farthing, Rebecca J.; Lale-Farjat, Shamar L. M. Frontiers in Molecular Biosciences, Vol. 7 https://doi.org/10.3389/fmolb.2020.605236	journal	December 2020
Optimization of the expression of the main protease from SARS-CoV-2 Rong, Yi; Zhang, Chaofeng; Gao, Wen-Chao Protein Expression and Purification, Vol. 203 https://doi.org/10.1016/j.pep.2022.106208	journal	March 2023