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

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

Journal Article · Sun Nov 12 23:00:00 EST 2023 · Communications Biology

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

Aniana, Annie ^[1]; ^[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.

View Accepted Manuscript (DOE)

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

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