摘要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 sub-lineages,it has maintained a prominent role ever since.The Nsp5 main protease(Mpro)of the Omicron virus is charac-terized by a single dominant mutation,P132H.Here we determined the X-ray crystal structures of the P132H mutant(or O-Mpro)as a free enzyme and in complex with the Mpro inhibitor,the alpha-ketoamide 13b-K,and we conducted enzy-mological,biophysical,as well as theoretical studies to characterize the O-Mpro.We found that O-Mpro has a similar overall structure and binding with 13b-K;however,it displays lower enzymatic activity and lower thermal stability compared to the WT-Mpro(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-Mpro dimer is destabilized relative to the WT-Mpro due to less favorable van der Waals interactions and backbone conformations in the individual protomers.All-atom continuous constant-pH mo-lecular dynamics(MD)simulations reveal that His132 and Glu240 display coupled titration.At pH 7,His132 is predom-inantly 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 Mpro 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 mu-tation sites.