title: Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS.
abstract: All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CL(pro)) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CL(pro) from other β-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CL(pro) is less efficient at processing a peptide substrate due to MERS-CoV 3CL(pro) being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CL(pro) enzymes are tightly associated dimers. Analytical ultracentrifugation studies support that MERS-CoV 3CL(pro) is a weakly associated dimer (Kd ∼52 μm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CL(pro) were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that MERS-CoV 3CL(pro) undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CL(pro) from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CL(pro) dimerization. Activation of MERS-CoV 3CL(pro) through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CL(pro) inhibitors as antiviral agents.
authors.affiliation: From the Departments of Biological Sciences and.
authors.forename: Sakshi
authors.initials: S
authors.lastname: Tomar
authors.affiliation: Chemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Melanie L
authors.initials: ML
authors.lastname: Johnston
authors.affiliation: From the Departments of Biological Sciences and.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: St John
authors.affiliation: Chemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Osswald
authors.affiliation: Chemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Prasanth R
authors.initials: PR
authors.lastname: Nyalapatla
authors.affiliation: the Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907, and.
authors.forename: Lake N
authors.initials: LN
authors.lastname: Paul
authors.affiliation: Chemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: the Departments of Pediatrics and Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Denison
authors.affiliation: From the Departments of Biological Sciences and Chemistry, Purdue University, West Lafayette, Indiana 47907, amesecar@purdue.edu.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Mesecar
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D000086782:Coronavirus 3C Proteases; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008024:Ligands; D065207:Middle East Respiratory Syndrome Coronavirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D057786:Peptidomimetics / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D055503:Protein Multimerization / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D008024:Ligands; D057786:Peptidomimetics; D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases
keywords: MERS-CoV 3CLpro; X-ray crystallography; analytical ultracentrifugation; enzyme inactivation; enzyme inhibitor; enzyme kinetics; ligand-induced dimerization; monomer-dimer equilibrium; viral protease; β-CoV