Omicron P132H of SARS-CoV-2 Mpro is an allosteric mutant involved in modulating the dynamics of catalytic site entry loop

Dynamic allostery without conformational changes is well established in CoV-2 main protease, Mpro, where distal sidechain dynamics and ligand binding have shown to affect the catalytic efficiency. Understanding this allosteric regulation of CoV-2 Mpro is essential for designing Mpro specific drugs. Also, in the context of viral variants/evolution, where mutation at a distal site may alter the catalytic site dynamics, hence enzymatic activity; a probable drug evading mechanism of Mpro. One of the predominant Mpro mutations of Omicron variant is Pro132His. Structurally, this mutation site is located ~22 Å away from the catalytic site. The solved crystal structure of this mutant in complex with inhibitors, as well as the reported catalytic efficiency didn’t show any difference with respect to the wild type. Thus, it is concluded to be non-allosteric. In this present work, based on the microsecond long Molecular Dynamics simulation of this Pro132His mutant and wild type, we show that Pro132His mutation affects the conformational equilibrium, with more population of conformational substates having open catalytic site, modulated by the dynamics of the catalytic site entry loop; implying the allosteric nature of this mutant. This population shift of open conformers is reflected in the increase in reported KM values of FRET assays, though its catalytic efficiency is comparable to that of wild type. Further, the structural analysis of microsecond simulation data indicates that rearrangement of hydrogen bonds between His132 and adjacent residues enhances the dynamics of the linker, which in turn is aggravated by the inherent dynamic flexibility of the catalytic pocket entry site. This inherent dynamics of catalytic pocket entry site is due to the fluctuating electrostatics brought out mainly by the charged residues of turns (Glu47, Asp48) and α2 helix (Glu55, Asp56, Arg60, Lys61) spanning the catalytic pocket entry site and base. This in turn results in the loss of key hydrogen bond between residues Met49-Gln189 and Asp187-Tyr54 of catalytic site, followed by the opening of the catalytic pocket entry site. Thus, Tyr54 could play possible role of being a gate keeper residue. Altered dynamics lead to loss of secondary structures corroborate well with the reported compromised thermal stability of this mutant. This study stands out as an example for how allosteric mutant can modulate the dynamics of protein, probably for its functional diversification.