A P3' methionine-to-phenylalanine substitution in the NSP6-7 substrate peptide alters SARS-CoV-2 M^pro dynamics and...

The SARS-CoV-2 main protease (M^pro) cleaves viral polyproteins at 11 sites to release nonstructural proteins required for replication, with site-specific sequence differences influencing processing efficiency. Among these substrates, NSP6-7 is consistently the slowest cleaved, while NSP4-5 is among the fastest. However, the mechanistic basis for slow NSP6-7 processing remains unclear. Here, we combined molecular dynamics (MD) simulations with DuProSense biosensor-based live-cell and in vitro assays to show that the P3' M is a key regulator of NSP6-7 binding, and cleavage by M^pro and M-to-F substitution increases its cleavage in living cells. MD simulations of dimeric M^pro bound to either NSP4-5 or NSP6-7 revealed increased flexibility and altered positioning of the C-terminal P2', P3', and P4' residues of NSP6-7 compared with NSP4-5. Guided by these observations, we individually replaced these NSP6-7 C-terminal residues with the corresponding residues from NSP4-5. BRET-based cleavage assays using DuProSense biosensor showed that the P3' M-to-F substitution, but not the P2' K-to-G or P4' S-to-R substitutions, increased NSP6-7 cleavage by M^pro in living cells, consistent with increased apparent substrate affinity in vitro. Comparison of structural dynamics of M^pro bound to NSP6-7(M7F) with NSP4-5 and NSP6-7 showed that the P3' substitution resulted in a dynamic profile more similar to NSP4-5 while introducing distinct NSP6-7(M7F)-specific effects, including enhanced correlated motions and reduced flexibility within C44-L50 residues in M^pro. Taken together, these results suggest that M7F mutation-induced enhanced cleavage of NSP6-7 arises from both restored and mutation-specific dynamic effects.