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

The monomeric catalytic domain (residues 1-199) of SARS-CoV-2 main protease (MPro) fused to 25 amino acids of its flanking nsp4 region mediates its autoprocessing at the nsp4-MPro junction. We report the catalytic activity and the dissociation constants of MPro 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 and MPro 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.