Distal Protein-Protein Interactions Contribute to SARS-CoV-2 Main Protease Substrate Binding and Nirmatrelvir Resistance

SARS-CoV-2 main protease, M ^pro , is responsible for the processing of the viral polyproteins into individual proteins, including the protease itself. M ^pro is a key target of anti-COVID-19 therapeutics such as nirmatrelvir (the active component of Paxlovid). Resistance mutants identified clinically and in viral passage assays contain a combination of active site mutations (e.g. E166V, E166A, L167F), which reduce inhibitor binding and enzymatic activity, and non-active site mutations (e.g. P252L, T21I, L50F), which restore the fitness of viral replication. Although the mechanism of resistance for the active site mutations is apparent, the role of the non-active site mutations in fitness rescue remains elusive. In this study, we use the model system of a M ^pro triple mutant (L50F/E166A/L167F) that confers not only nirmatrelvir drug resistance but also a similar fitness of replication compared to the wild-type both in vitro and in vivo. By comparing peptide and full-length M ^pro protein as substrates, we demonstrate that the binding of M ^pro substrate involves more than residues in the active site. In particular, L50F and other non-active site mutations can enhance the M ^pro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of M ^pro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying M ^pro and substrate interactions, and offers important insights into M ^pro function, resistance development, and inhibitor design.