Distal protein-protein interactions contribute to nirmatrelvir resistance

Abstract

SARS-CoV-2 main protease, M^pro, is responsible for processing 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. To probe the role of the non-active site mutations in fitness rescue, here we use an M^pro triple mutant (L50F/E166A/L167F) that confers nirmatrelvir drug resistance with a viral fitness level similar to the wild-type. 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. Particularly, 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.

Fig. 1. Characterization of M^pro mutants reveals differences between protein and peptide nsp5-6 substrates.

a Overview of the M^pro dimer (cyan/orange) showing nirmatrelvir (yellow) bound in the active site and the locations of mutations of interest in the active site (red spheres, e.g., E166/L167) and distal to the active site (blue spheres, e.g., L50) (PDB 8DCZ). M^pro is also named nsp5 on viral polyproteins. b M^pro self-cleavage at the C-terminus. E: M^pro as enzyme, S: M^pro as substrate. The polyprotein chain after M^pro is represented by the curved line. c Schematic of how the fluorescence gel-based assay functions. A protein substrate is constructed by using the catalytically inactive C145A M^pro conjugated with a bacterial protein PBP3, serving as a reporter for quantification purposes after it reacts with the fluorescent inhibitor Bocillin. A schematic image for the assay was created in BioRender. Kohaal, N. (2025) https://BioRender.com/n42a748. d Rate of activity for the C145A M^pro-PBP3 and the C145A/L50F M^pro-PBP3 substrates, in comparison to the peptide substrates. The rate of activity for the C145A/T21I M^pro-PBP3 is shown in Supplementary Fig. 1a. e Cleavage reaction of C145A-PBP3 by multiple M^pro mutants. The cleavage reaction of C145A/L50F M^pro-PBP3 by the same M^pro mutants is shown in Supplementary Fig. 1b. Source data for Fig. 1d and e are provided as a Source Data file. Rates are the average of two replicates.

Fig. 2. M^pro L50F/E166A/L167F triple mutant crystal structure showing L50F mutation at the protein-protein interface.

a M^pro triple mutant dimer of a dimer (dark green/green, magenta/salmon), showing P1–P6 residues (magenta stick) of the C-terminus from the substrate dimer (post cleavage, a.k.a, product) bound in the active site of the enzyme dimer (green). Zoomed-in view shows the interactions of the enzyme F50 (green) within the substrate hydrophobic pocket (magenta). Substrate residues are noted in red text. b Close-up view of the binding pose of P1–P6 bound in the triple mutant active site (substrate shown in magenta and enzyme shown in green). The N-terminus from an adjacent protomer is noted in orange. Substrate residues are noted in red text. c Movement of the 166–168 backbone in the triple mutant with P1–P6 bound in the active site (magenta/green) vs. M^pro C145A with nsp5/6 substrate bound (cyan/light purple) (PDB 7MB5). Substrate residues are noted in red text, and the N-terminus from an adjacent monomer is noted in orange. d Binding pose of nirmatrelvir (white, PBD 8DCZ) superimposed into the triple mutant binding pocket. The N-terminus from an adjacent protomer is noted in orange.

Fig. 3. M^pro L50F single mutant crystal structure with unique substrate binding mode.

a L50F single mutant dimer of a dimer (yellow/wheat, blue/light blue), showing P1–P6 (light blue) of the C-terminus bound in the chain B (wheat) active site. The zoomed-in view shows the interactions of the enzyme F50 with the substrate hydrophobic pocket. A view of the chain A interactions can be found in Supplementary Fig. 4. Mutation is noted in red text. b Binding of P1–P6 in the L50F chain B active site. The substrate protomer is shown in light blue, and the enzyme protomer is shown in wheat. The mutation is noted in red text. c Comparison of the binding modes of P1–P6 in the L50F single mutant (light blue/wheat) vs. the L50F/E166A/L167F triple mutant (magenta/green). Mutations are noted in red text.