This article was originally published here
J Biol Chem. May 11, 2022:102023. doi: 10.1016/j.jbc.2022.102023. Online ahead of print.
Type 3C protease (3CLpro) is one of two proteases that process and release functional viral proteins essential for the maturation and infectivity of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19. 3CLpro has been suggested to be catalytically active as a dimer, making the dimerization interface a target for antiviral development. Guided by structural analysis, here we introduced single nine-residue amino acid substitutions at three key sites of the dimer interface to assess their impact on dimerization and activity. We show that at site 1, alanine substitution of S1 or E166 increased 2-fold or reduced relative activity, respectively. At site 2, alanine substitution of S10 or E14 eliminated activity, while K12A exhibited approximately 60% relative activity. At site 3, alanine substitution of R4, E290, or Q299 eliminated activity, while S139A exhibited 46% relative activity. We further found that the oligomerization states of the dimer interface mutants varied; inactive mutants R4A, R4Q, S10A/C, E14A/D/Q/S, E290A and Q299A/E were present as dimers, demonstrating that dimerization is not indicative of catalytically active 3CLpro. Moreover, present mainly as monomers, K12A displayed residual activity, which could be attributed to the remarkable amount of dimers present. Finally, differential scanning calorimetry did not reveal a direct relationship between the thermodynamic stability of mutants with oligomerization or catalytic activity. These results provide information on two allosteric sites, R4/E290 and S10/E14, that may favor the design of antiviral compounds that target the dimer interface rather than the active site of SARS-CoV-2 3CLpro.