Michaelis-like complex of SARS-CoV-2 main protease visualized by roomerature X-ray crystallography

Daniel W. Kneller, Qiu Zhang, Leighton Coates, John M. Louis, Andrey Kovalevsky

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

SARS-CoV-2 emerged at the end of 2019 to cause an unprecedented pandemic of the deadly respiratory disease COVID-19 that continues to date. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and is therefore an important drug target. Understanding the catalytic mechanism of Mpro, a cysteine protease with a catalytic site comprising the noncanonical Cys145-His41 dyad, can help in guiding drug design. Here, a 2.0 Å resolution roomerature X-ray crystal structure is reported of a Michaelis-like complex of Mpro harboring a single inactivating mutation C145A bound to the octapeptide Ac-SAVLQSGF-CONH2 corresponding to the nsp4/nsp5 autocleavage site. The peptide substrate is unambiguously defined in subsites S5 to S3′ by strong electron density. Superposition of the Michaelis-like complex with the neutron structure of substrate-free Mpro demonstrates that the catalytic site is inherently pre-organized for catalysis prior to substrate binding. Induced fit to the substrate is driven by P1 Gln binding in the predetermined subsite S1 and rearrangement of subsite S2 to accommodate P2 Leu. The Michaelis-like complex structure is ideal for in silico modeling of the SARS-CoV-2 Mpro catalytic mechanism.

Original languageEnglish
Pages (from-to)973-979
Number of pages7
JournalIUCrJ
Volume8
DOIs
StatePublished - Nov 1 2021

Funding

This research used resources at Oak Ridge National Laboratory’s Center for Structural Molecular Biology (CSMB), a DOE Office of Science User Facility, supported by the Office of Biological and Environmental Research. This research used resources of the Spallation Neutron Source Second Target Station Project at Oak Ridge National Laboratory (ORNL). ORNL is managed by UT-Battelle LLC for DOE’s Office of Science, the single largest supporter of basic research in the physical sciences in the United States. We thank Dr Hugh M. O’Neill from ORNL for assistance during expression and purification of the protein. We thank Annie Aniana for excellent technical assistance, John Lloyd and the NIDDK core facility for mass spectrometry. Author contributions were as follows. DWK, LC and AK conceived the study, QZ and JML constructed, expressed and purified Mpro/C145A, DWK crystallized the protein and DWK and AK collected and processed the X-ray data and refined the structure. The manuscript was written with the contribution of all authors. All authors have given approval to the final version of the manuscript. This research was supported by the DOE Office of Science through the National Virtual Biotechnology Laboratory (NVBL), a consortium of DOE national laboratories focused on response to COVID-19, with funding provided by the Coronavirus CARES Act. This work was also supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, NIH.

FundersFunder number
National Virtual Biotechnology Laboratory
Oak Ridge National Laboratory
National Institutes of Health
U.S. Department of Energy
National Institute of Diabetes and Digestive and Kidney Diseases
Office of Science
Biological and Environmental Research
Canadian Society for Molecular Biosciences

    Keywords

    • 3CL protease
    • C145A mutant
    • Catalytic mechanism
    • Enzyme-substrate complex
    • Main protease
    • Roomerature X-ray crystallography
    • SARS-CoV-2

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