The C117D oxidation mimic reveals the monomeric structure of SARS-CoV-2 main protease.

Abstract

The SARS-CoV-2 main protease (M^pro) is essential for viral replication and functions as a homodimer, with dimerization being critical for catalytic activity. M^pro contains an unusually high number of cysteine residues. Among these, C117 and the catalytic nucleophile C145 can form a reversible disulfide bond under oxidative conditions. To investigate the structural and functional consequences of irreversible oxidation of these residues, we generated oxidation mimics by substituting these residues with aspartate (C117D and C145D), to mimic the sulfinic acid oxidation state. Kinetic assays revealed that both variants are catalytically inactive, with C117D exhibiting at least 100-fold lower activity than wild-type (WT) M^pro. Small-angle X-ray scattering (SAXS) and differential scanning fluorimetry (DSF) demonstrated that C117D adopts a monomeric, destabilized state in solution, whereas C145D retains a dimeric conformation similar to WT. Crystallographic analysis of C117D revealed a dramatic rearrangement of domain III, involving a ~40° rotation relative to domains I and II, and disorder in the N- and C-terminal regions, disrupting the canonical dimerization interface. Local structural changes propagated from the C117D site to the active site, including an unwound oxyanion loop that provides structural evidence for the observed inactivity. Rescue of the stable, dimeric state for C117D was achieved through formation of the covalent C117D-GC376 complex. These findings establish a high-resolution structure of monomeric full-length SARS-CoV-2 M^pro and underscore a critical role of C117 in maintaining dimerization and enzymatic function. Furthermore, the unique monomeric domain II-III interface present in the monomeric form may offer opportunities for allosteric inhibitor design targeting M^pro dimerization.