SARS-CoV-2 polyprotein substrate regulates the stepwise M^pro cleavage reaction

The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease M^pro to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. M^pro cleaves polyproteins in a defined order, but how M^pro and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound M^pro. Here, we present the cryo-EM structures of SARS-CoV-2 M^pro in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that M^pro interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of M^pro highlights the flexible nature of the active site region of M^pro, which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of M^pro in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by M^pro.