Unexpected single ligand occupancy and negative cooperativity in the SARS-CoV-2 Main protease

Many homodimeric enzymes tune their function by exploiting either negative or positive cooperativity between subunits. In the SARS-CoV-2 Main protease (Mpro) homodimer, the latter has been suggested by symmetry in most of the 500 reported protease/ligand complex structures solved by macromolecular crystallography. Here we apply the latter to both covalent and non-covalent ligands in complex with Mpro. Strikingly, our experiments show that occupation of both active sites of the dimer originates from an excess of ligands. Indeed, co-crystals obtained using a 1:1 ligand/protomer stoichiometry leads to single occupation only. The empty binding site exhibits a catalytic-inactive geometry in solution, as suggested by molecular dynamics simulations. Thus, Mpro operates through negative cooperativity, with asymmetric activity of the catalytic sites. This allows it to function with a wide range of substrate concentrations, making it resistant to saturation and potentially difficult to shut down - all properties advantageous for the virus’ adaptability and resistance.