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

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 center dot 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 center dot 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.