Why is the Omicron main protease of SARS-CoV-2 less stable than its wild-type counterpart? A crystallographic, biophysical, and theoretical study of the free enzyme and its complex with inhibitor 13b-K.

During the continuing evolution of SARS-CoV-2, the Omicron variant of concern emerged in the second half of 2021 and has been dominant since November that year. Along with its sublineages, it has maintained a prominent role ever since. The Nsp5 main protease (M) 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) as free enzyme and in complex with the M inhibitor, the alpha-ketoamide , and we conducted enzymology, biophysical as well as theoretical studies to characterize the O-M. We found that O-M has a similar overall structure and binding with ; however, it displays lower enzymatic activity and lower thermal stability compared to the WT-M (with "WT" referring to the original Wuhan-1 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. The empirical folding free energy calculations suggest that the O-M dimer is destabilized relative to the WT-M due to the less favorable van der Waals interactions and backbone conformation in the individual protomers. The 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 mutations, we also determined crystal structures of the relatively frequent P132H+T169S double mutant but found little evidence for a correlation between the two sites.