Probing the Dynamic Structure–Function and Structure-Free Energy Relationships of the Coronavirus Main Protease with Biodynamics Theory

Abstract

The SARS-CoV-2 main protease (M pro ) is of major interest as an antiviral drug target. Structure-based virtual screening efforts, fueled by a growing list of apo and inhibitor-bound SARS-CoV/CoV-2 M pro crystal structures, are underway in many laboratories. However, little is known about the dynamic enzyme mechanism, which is needed to inform both assay development and structure-based inhibitor design. Here, we apply biodynamics theory to characterize the structural dynamics of substrate-induced M pro activation under nonequilibrium conditions . The catalytic cycle is governed by concerted dynamic structural rearrangements of domain 3 and the m-shaped loop (residues 132–147) on which Cys145 (comprising the thiolate nucleophile and half of the oxyanion hole) and Gly143 (comprising the second half of the oxyanion hole) reside. In particular, we observed the following: (1) Domain 3 undergoes dynamic rigid-body rotation about the domain 2–3 linker, alternately visiting two primary conformational states (denoted as M 1 pro ↔ M 2 pro ); (2) The Gly143-containing crest of the m-shaped loop undergoes up and down translations caused by conformational changes within the rising stem of the loop (Lys137–Asn142) in response to domain 3 rotation and dimerization (denoted as M 1/down pro ↔ 2·M 2/up pro ) (noting that the Cys145-containing crest is fixed in the up position). We propose that substrates associate to the M 1/down pro state, which promotes the M 2/down pro state, dimerization (denoted as 2·M 2/up pro –substrate), and catalysis. Here, we explore the state transitions of M pro under nonequilibrium conditions, the mechanisms by which they are powered, and the implications thereof for efficacious inhibition under in vivo conditions.

Dynamic Profiling of β-Coronavirus 3CL Mpro Protease Ligand-Binding Sites

“…Most MD studies have focused only on the substrate-binding site of the M pro enzyme. 51 − 53 Other computational studies have looked into identifying novel pockets and investigating allostery. 54 , 55 However, these studies are limited in comparing dynamics with the vast crystallographic data available on ortho- and allosteric ligand-binding sites across β-CoV homologs.…”

A higher flexibility at the SARS-CoV-2 main protease active site compared to SARS-CoV and its potentialities for new inhibitor virtual screening targeting multi-conformers

“…Moreover, substrate binding to monomers can favor the dimeric state (Cheng et al., 2010 ). Even though only dimers are enzymatically active, some authors propose M pro catalytic cycle models in which up (activated) and down (inactivated) states can be found at equilibrium in monomers (see figure 8 in Cheng et al., 2010 and figure 29 in Wan et al., 2020 ). But, only up monomers are capable of dimerization, especially those with bound substrates.…”

Structural dynamics of the β-coronavirus Mpro protease ligand binding sites

“…Most molecular dynamics studies have focused only on the substrate binding site of the M pro enzyme. [50][51][52] Other computational studies have looked into identifying novel pockets and investigating allostery. 53,54 However, these studies are limited in comparing dynamics with the vast crystallographic data available on ortho-and allosteric ligand binding sites across β-coronavirus homologs.…”