Recognition of Divergent Viral Substrates by the SARS-CoV-2 Main Protease

MacDonald, Elizabeth A.; Frey, Gary; Namchuk, Mark N.; Harrison, Stephen C.; Hinshaw, Stephen M.; Windsor, Ian W.

[Image: see text] The main protease (M(pro)) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. M(pro) is conserved among coronaviruses and distinct from human proteases. Viral replication depends on the cleavage of the viral polyprotein at multiple sites. We present crystal structures of SARS-CoV-2 M(pro) bound to two viral substrate peptides. The structures show how M(pro) recognizes distinct substrates and how subtle changes in substrate accommodation can drive large changes in catalytic efficiency. One peptide, constituting the junction between viral nonstructural proteins 8 and 9 (nsp8/9), has P1′ and P2′ residues that are unique among the SARS-CoV-2 M(pro) cleavage sites but conserved among homologous junctions in coronaviruses. M(pro) cleaves nsp8/9 inefficiently, and amino acid substitutions at P1′ or P2′ can enhance catalysis. Visualization of M(pro) with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.