Kinetic control of MERS‐CoV 3CL ^pro is mediated by non‐conserved residues distal to the active site

Middle‐East Respiratory Syndrome (MERS) Coronavirus is a re‐emergent zoonotic pathogen with a 30% fatality rate in humans. The MERS positive‐sense single‐stranded RNA genome is translated into two polyproteins that must be processed by two viral cysteine proteases (3CL pro and PL pro ) into 16 non‐structural proteins (nsp) before viral replication can occur. Attempts to drug either of these cysteine proteases have proven difficult. MERS 3CL pro functions as dimer and is unique among all of the coronavirus 3CL pro enzymes studied to date because competitive inhibitors activate the wild‐type MERS 3CL pro enzyme at low inhibitor concentrations and inhibit at higher concentrations. This mechanism of substrate/inhibitor‐induced dimerization seems to be mediated by non‐conserved residues in structural regions distal to the catalytic site. We have identified unique residues that are involved in the inhibitor‐induced dimerization mechanism and we show that these ‘competitive’ inhibitors activate and inhibit in a dose‐dependent manner. In an attempt to provide a structural basis for this mechanism, we crystallized the apo and inhibitor‐bound forms of this enzyme and diffracted them to 1.5 – 2.2 Å. We also show that residues in distinct structural regions synergize to modulate the intrinsic kinetic parameters of this family of enzymes. These parameters include increased turnover (k cat ) and a lower dimer dissociation constant (K D ). Additionally, a rapid‐equilibrium kinetic mechanism was developed to model the kinetic response of MERS 3CLpro to inhibitors that act via substrate/inhibitor induced activation/inhibition mechanisms. Support or Funding Information This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health , Department of Health and Human Services, under Contracts No. HHSN272200700058C, HHSN272201200026C, and HHSN272201700060C. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357.Use of the Lilly Research Laboratories Collaborative Access Team (LRL‐CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility.