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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., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The monomeric catalytic domain (residues 1-199) of SARS-CoV-2 main protease (MPro 1-199 ) fused to 25 amino acids of its flanking nsp4 region mediates its autoprocessing at the nsp4-MPro 1-199 junction. We report the catalytic activity and the dissociation constants of MPro 1-199 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 MPro WT and MPro 1-199 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., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)