Why is the Omicron main protease of SARS-CoV-2 less stable than its wild-type counterpart? A crystallographic, biophysical, and theoretical study

During the continuing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the Omicron variant of concern emerged in the second half of 2021 and has been dominant since November of that year. Along with its sublineages, it has maintained a prominent role ever since. The Nsp5 main protease (M^pro) of the Omicron virus is characterized by a single dominant mutation, P132H. Here we determined the X-ray crystal structures of the P132H mutant (or O-M^pro) as a free enzyme and in complex with the M^pro inhibitor, the alpha-ketoamide 13b-K, and we conducted enzymological, biophysical, as well as theoretical studies to characterize the O-M^pro. We found that O-M^pro has a similar overall structure and binding with 13b-K; however, it displays lower enzymatic activity and lower thermal stability compared to the WT-M^pro (with “WT” referring to the prototype strain). Intriguingly, the imidazole ring of His132 and the carboxylate plane of Glu240 are in a stacked configuration in the X-ray structures determined here. Empirical folding free energy calculations suggest that the O-M^pro dimer is destabilized relative to the WT-M^pro due to less favorable van der Waals interactions and backbone conformations in the individual protomers. All-atom continuous constant-pH molecular dynamics (MD) simulations reveal that His132 and Glu240 display coupled titration. At pH 7, His132 is predominantly neutral and in a stacked configuration with respect to Glu240 which is charged. In order to examine whether the Omicron mutation eases the emergence of further M^pro mutations, we also analyzed the P132H+T169S double mutant, which is characteristic of the BA.1.1.2 lineage. However, we found little evidence of a correlation between the two mutation sites.