The Conformational Space of the SARS-CoV-2 Main Protease Active Site Loops is Determined by Ligand Binding and Interprotomer Allostery

The main protease (MPro) of SARS-CoV-2 is essential for viral replication and is, therefore, an important drug target. Here, we investigate two flexible loops in MPro that play a role in catalysis. Using all-atom molecular dynamics simulations, we analyze the structural ensemble of MPro in an apo state and substrate-bound state. We find that the flexible loops can adopt open, intermediate (partly open) and closed conformations in solution, which differs from the partially closed state observed in crystal structures of MPro. When the loops are in closed or intermediate states, the catalytic residues are more likely to be in close proximity, which is crucial for catalysis. Additionally, we find that substrate binding to one protomer of the homodimer increases the frequency of intermediate states in the bound protomer, while also affecting the structural propensity of the apo protomer's flexible loops. Using dynamic network analysis, we identify multiple allosteric pathways connecting the two active sites of the homodimer. Common to these pathways is an allosteric hotspot involving the N-terminus, a critical region that comprises part of the binding pocket. Taken together, the results of our simulation study provide detailed insight into the relationships between the flexible loops and substrate binding in a prime drug target for COVID-19.