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 (Mpro) 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 Mpro active site is crucial for catalysis and this interaction has been employed to inform inhibitor design; however, how Mpro 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 Mpro 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 Mpro 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 Mpro 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.