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

Abstract

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 Mproactive site is crucial for catalysis and this interaction has been employed to inform inhibitor design; however, how Mprodynamically 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 Mproto 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 productivegaucheconformation to the non-productivetransconformation. Importantly, our findings indicate that Mproexhibits 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 Mprotowards 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.

Signal Propagation in the ATPase Domain of Mycobacterium tuberculosis DNA Gyrase from Dynamical-Nonequilibrium Molecular Dynamics Simulations

“…This is a powerful approach because it allows for direct computation of the time-dependent response of the system to a perturbation while assessing the statistical significance of that response. We have previously applied the D-NEMD methodology, using nonequilibrium simulations of ≤5 ns, to investigate several systems, including: identifying allosteric effects in the SARS-CoV-2 spike protein; removing ligands from the SARS-CoV-2 main protease; showing cooperativity and allosteric effects involving positions associated with drug resistance; identifying allosteric networks in class A β-lactamase enzymes; identifying a general mechanism of signal propagation in nicotinic acetylcholine receptors; and the ATP hydrolysis mechanism of the ABC transporter NBD dimer. ,− …”

“…The vectors show the amplitude of the structural responses to the perturbation and the average direction of these motions (Figures and S11). ,, …”