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 Å resolution, in its acyl-enzyme intermediate state with the native C-terminal autocleavage sequence at 1.95 Å resolution and in its product bound state at 2.0 Å 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.
【저자키워드】 SARS-CoV-2, Drug discovery, X-ray crystallography, Enzyme mechanisms, 【초록키워드】 COVID-19, Mutation, Antiviral, molecular docking, protease, SARS-CoV-2 main protease, coronavirus 2, antiviral drug, pathogen, viral replication, therapeutic, cleavage, nonstructural protein, mutant, respiratory, Critical, mechanism, binding, conformational change, maturation, cysteine protease, M pro, sequence, wild-type, polyproteins, feature, event, autocleavage, responsible, identify, subsequent, the disease, determine, occur, cause, representing, C-terminal, catalytic, replicase polyprotein, X-ray crystallographic structure, 【제목키워드】 protease, physiological, wild-type SARS-CoV-2, C-terminal,