Crystallographic structure of wild-type SARS-CoV-2 main protease acyl-enzyme intermediate with physiological C-terminal autoprocessing site

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes the disease COVID-19, produces replicase polyproteins 1a and 1ab that contain, respectively, 11 or 16 nonstructural proteins (nsp). Nsp5 is the main protease (M-pro) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for subsequent viral assembly and maturation. We have determined X-ray crystallographic structures of this cysteine protease in its wild-type free active site state at 1.8 angstrom resolution, in its acyl-enzyme intermediate state with the native C-terminal autocleavage sequence at 1.95 angstrom resolution and in its product bound state at 2.0 angstrom resolution by employing an active site mutation (C145A). We characterize the stereochemical features of the acyl-enzyme intermediate including critical hydrogen bonding distances underlying catalysis in the Cys/His dyad and oxyanion hole. We also identify a highly ordered water molecule in a position compatible for a role as the deacylating nucleophile in the catalytic mechanism and characterize the binding groove conformational changes and dimerization interface that occur upon formation of the acyl-enzyme. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for future antiviral therapeutic development including revised molecular docking strategies based on M-pro inhibition. The SARS-CoV-2 main protease (M-pro) is one of two cysteine proteases essential for viral replication. Here, the authors determine the crystal structure of an M-pro acyl intermediate with its native C-terminal autocleavage sequence and the structure of a product bound active site mutant (C145A), which are of interest for antiviral drug development.