Substrate recognition and selectivity in SARS-CoV-2 main protease: Unveiling the role of subsite interactions through dynamical nonequilibrium molecular dynamics simulations

The main protease (M pro ) of the SARS-CoV-2 coronavirus employs a cysteine-histidine dyad in its active site to catalyse hydrolysis of the viral polyproteins. It is well established that binding of the substrate P1-Gln in the S1 subsite of M pro active site is crucial for catalysis and this interaction has been employed to inform inhibitor design; however, how M pro dynamically recognises and responds to substrate binding remains difficult to probe by experimental methods. We thus employed the dynamical nonequilibrium molecular dynamics (D-NEMD) approach to probe the response of M pro to systematic substrate variations. The results emphasise the importance of P1-Gln for initiating a productive enzymatic reaction. Specifically, substituting P1-Gln with alanine disrupts the conformations of the Cys145 and His41 dyad, causing Cys145 to transition from the productive gauche conformation to the non-productive trans conformation. Importantly, our findings indicate that M pro exhibits dynamic responses to substrate binding and likely to substrate-mimicking inhibitors within each of the S4-S2′ subsites. The results inform on the substrate selectivity requirements and shed light on the observed variations in hydrolytic efficiencies of M pro towards different substrates. Some interactions between substrate residues and enzyme subsites involve more induced fit than others, implying that differences in functional group flexibility may optimise the binding of a substrate or inhibitor in a particular subsite.