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

02 June 2023, Version 1


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.


Main protease
ligand occupancy
negative cooperativity
macromolecular crystallography
molecular dynamics
inhibitory activity

Supplementary materials

Supplementary information for Unexpected single ligand occupancy and negative cooperativity in the SARS-CoV-2 Main protease
Figures, tables, charts and some supplementary information


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