Autoprocessing and oxyanion loop reorganization upon GC373 and nirmatrelvir binding of monomeric SARS-CoV-2 main protease catalytic domain

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

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

The monomeric catalytic domain (residues 1–199) of SARS-CoV-2 main protease (MPro1-199) fused to 25 amino acids of its flanking nsp4 region mediates its autoprocessing at the nsp4-MPro1-199 junction. We report the catalytic activity and the dissociation constants of MPro1-199 and its analogs with the covalent inhibitors GC373 and nirmatrelvir (NMV), and the estimated monomer-dimer equilibrium constants of these complexes. Mass spectrometry indicates the presence of the accumulated adduct of NMV bound to MProWT and MPro1-199 and not of GC373. A room temperature crystal structure reveals a native-like fold of the catalytic domain with an unwound oxyanion loop (E state). In contrast, the structure of a covalent complex of the catalytic domain-GC373 or NMV shows an oxyanion loop conformation (E* state) resembling the full-length mature dimer. These results suggest that the E-E* equilibrium modulates autoprocessing of the main protease when converting from a monomeric polyprotein precursor to the mature dimer.

Original languageEnglish
Article number976
JournalCommunications Biology
Volume5
Issue number1
DOIs
StatePublished - Dec 2022

Funding

This research used resources at the Spallation Neutron Source, and the High Flux Isotope Reactor, which are DOE Office of Science User Facilities operated by the Oak Ridge National Laboratory. The Office of Biological and Environmental Research supported research at ORNL’s Center for Structural Molecular Biology (CSMB), a DOE Office of Science User Facility. 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 are grateful to Dr. Rolf Hilgenfeld and his staff for providing us with an expression plasmid to get us initially started on the main protease work. We thank John Lloyd and the NIDDK mass spectrometry core facility. This work was supported by the Intramural Research Program of National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH. This research used resources at the Spallation Neutron Source, and the High Flux Isotope Reactor, which are DOE Office of Science User Facilities operated by the Oak Ridge National Laboratory. The Office of Biological and Environmental Research supported research at ORNL’s Center for Structural Molecular Biology (CSMB), a DOE Office of Science User Facility. 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 are grateful to Dr. Rolf Hilgenfeld and his staff for providing us with an expression plasmid to get us initially started on the main protease work. We thank John Lloyd and the NIDDK mass spectrometry core facility. This work was supported by the Intramural Research Program of National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH.

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