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. 2015 Aug 7;290(32):19403-22.
doi: 10.1074/jbc.M115.651463. Epub 2015 Jun 8.

Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS

Sakshi Tomar  1 , Melanie L Johnston  2 , Sarah E St John  1 , Heather L Osswald  2 , Prasanth R Nyalapatla  2 , Lake N Paul  3 , Arun K Ghosh  2 , Mark R Denison  4 , Andrew D Mesecar  5
Affiliations

Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS

Sakshi Tomar et al. J Biol Chem. .

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.

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.

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Figures

FIGURE 1.
FIGURE 1.
Comparison of enzymatic efficiencies (kcat/Km) of 3CLpro enzymes from different CoVs. A, rates for the enzymatic activity, normalized to the total enzyme concentration, are plotted as a function of varying substrate concentrations. Total concentration of each enzyme in the final reaction is as follows: MERS-CoV 3CLpro at 1 μm; SARS-CoV 3CLpro at 100 nm; HKU5-CoV 3CLpro at 250 nm; and HKU4-CoV 3CLpro at 200 nm. Slope of the line represents the apparent value of kcat/Km. Error bars represent the standard deviation for triplicate data. B, *, apparent value of kcat/Km for the nonsaturable substrate, calculated as the slope of the linear plot from panel A.
FIGURE 2.
FIGURE 2.
Dependence of the enzymatic activity of MERS-CoV, HKU4-CoV, HKU5-CoV, and SARS-CoV 3CLpro on the total enzyme concentration. A, kinetic response of each CoV 3CLpro to increasing enzyme concentration is plotted along with the resulting fit of the data to Equation 2. Resulting values for the apparent turnover number, kcat, and the monomer-dimer equilibrium constant, Kd, are shown in Table 2. Final enzyme concentrations varied over the concentration ranges of 2 μm to 100 nm for MERS-CoV 3CLpro, 500 to 10 nm for SARS-CoV 3CLpro, 250 to 0.6 nm for HKU5-CoV 3CLpro, and 200 to 10 nm for HKU4-CoV 3CLpro. Final substrate concentration was fixed at 2 μm. Experiments were done in triplicate. Error bars represent the standard deviation for triplicate data. Shaded box represents the data that are plotted in B. B, enlarged view of the fitted data at low total enzyme concentrations, marked in shaded box in A, illustrating the nonlinear dependence of enzymatic activity on the total concentrations of 3CLpro from SARS-CoV, HKU5-CoV, and HKU4-CoV.
FIGURE 3.
FIGURE 3.
Progress curves for the MERS-CoV 3CLpro-catalyzed reaction in the presence of compound 6. Time-dependent hydrolysis of 1 μm substrate catalyzed by 500 nm MERS-CoV 3CLpro was measured over a time period of 70 min and at fixed variable concentrations of compound 6 ranging from 0 to 50 μm. Values for the inactivation kinetic parameters kinact, t½∞, and KI were calculated by fitting the progress curve data to Equations 4 and 5. Chemical structure of compound 6 is shown in the inset.
FIGURE 4.
FIGURE 4.
AUC-SV analyses of ligand-induced dimerization of MERS-CoV 3CLpro. A, sedimentation coefficient distribution for varying concentrations of MERS-CoV 3CLpro (4.1 to 23 μm) with sedimentation coefficient values of 2.9S and 3.9S for the monomer and the dimer, respectively. The best fit value for AUC-SV-calculated Kd is 52 ± 5 μm. B, sedimentation coefficient distribution of MERS-CoV 3CLpro (25 μm) in the presence of different stoichiometric ratios of compound 6 (25, 50, and 100 μm). C, sedimentation coefficient distribution of MERS-CoV 3CLpro (25 μm) in the presence of different stoichiometric ratios of compound 10 (25, 50, and 100 μm). A significant shift in the 2.9S peak (monomer) to a 4.1S peak (dimer) is detected upon addition of increasing concentrations of compounds 6 and 10.
FIGURE 5.
FIGURE 5.
Activation of MERS-CoV 3CLpro via ligand-induced dimerization. A, enzymatic activity of 0.5, 1.0, and 2.0 μm MERS-CoV 3CLpro was measured in the absence and presence of varying concentrations of compound 10. Substrate concentration was fixed at 2.0 μm. % activity, normalized to zero inhibitor enzymatic activity, was plotted as a function of increasing inhibitor concentrations. Error bars represent the standard deviation for triplicate data. Increase in enzymatic activity (highlighted in cyan-shaded box) is observed in the presence of low concentrations of compound 10. Inhibition of enzymatic activity is observed at higher inhibitor concentrations (highlighted in yellow-shaded box). B, kinetic model describing the equilibrium between different species of MERS-CoV 3CLpro that are formed in the absence (blue box) and presence (green box) of a ligand is shown. Based on the AUC-calculated Kd value of ∼ 52 μm, MERS-CoV 3CLpro primarily exists as a monomer in solution in the absence of a ligand. Upon ligand binding (inhibitor I in our case) to the monomer, the monomer-dimer equilibrium shifts toward dimer formation. Next, under lower inhibitor concentrations (cyan-shaded box), substrate (S) binds in the second active site and catalysis takes place. However, under higher inhibitor concentrations (yellow-shaded box), inhibitor directly competes with the substrate for the second active site, and inhibition of the enzymatic activity is observed.
FIGURE 6.
FIGURE 6.
X-ray crystal structure of MERS-CoV 3CLpro in complex with inhibitors. A, solvent-accessible surface (gray-shaded surface) of MERS-CoV 3CLpro and compound 6 complex. Compound 6 is displayed in ball and stick model with atoms colored as follows: carbons (orange), nitrogens (blue), and oxygens (red). Electron density associated with compound 6 is shown as an Fo − Fc electron density difference map contoured to 3σ (green mesh). Substrate binding pockets S4-S′1 are labeled, where asterisk indicates the electrophilic carbon of compound 6 that forms a C–S covalent bond with the active site cysteine Cys-148. B, MERS-CoV 3CLpro and compound 6 complex with the MERS-CoV 3CLpro backbone represented as a ribbon model and relevant amino acids that interact with compound 6 represented as ball and sticks. MERS-CoV 3CLpro carbon atoms are colored blue, and compound 6 carbon atoms are colored orange. Nitrogen atoms are colored blue, and oxygen atoms are colored red. Catalytic residues Cys-148 and His-41 are also shown. Hydrogen bonds are depicted as red dashed lines. C, sequence logos showing amino acid conservation for the 11 polyprotein cleavage sites of different 3CLpro enzymes (MERS-CoV, HKU5-CoV, HKU4-CoV, and SARS-CoV), generated using the WebLogo server (63). Residues P2-P′1 are shown. Height of each letter corresponds to the amino acid conservation at that position. D, solvent-accessible surface (gray-shaded surface) of MERS-CoV 3CLpro and compound 11 complex. Compound 11 is displayed in ball and stick model. Electron density associated with compound 11 is shown as a 2Fo − Fc electron density difference map contoured to 1.5σ (green mesh). Functional groups of compound 11 with their corresponding binding pockets are highlighted in yellow, green, and blue ellipses. Chemical structure of compound 11 is shown in the inset. E, interactions between MERS-CoV 3CLpro and compound 11 are illustrated. Catalytic residues Cys-148 and His-41 are also shown. Hydrogen bonds are depicted as red dashed lines.
FIGURE 7.
FIGURE 7.
Comparison of x-ray crystal structures of 3CLpro dimers from MERS-CoV, HKU4-CoV, and SARS-CoV. A, superposition of dimers of MERS-CoV 3CLpro (pink color), HKU4-CoV 3CLpro (yellow color, PDB code 2YNB), and SARS-CoV 3CLpro (blue color, PDB code 2ALV). For SARS-CoV 3CLpro, residues Arg-4 and Ser-123 from monomer A, and residues Gln-127, Lys-137, Glu-290, and Met-298 from monomer B are represented as spheres. B, for SARS-CoV 3CLpro, interactions between the side chain of Arg-4 from monomer A and Gln-127, Glu-290, and Lys-137 residues from monomer B are shown. The corresponding residues in MERS-CoV 3CLpro and HKU4-CoV 3CLpro are Val-4 in monomer A and Glu-290 in monomer B, which do not interact at the dimer interface. C, for SARS-CoV 3CLpro, Ser-123 from monomer A engages in hydrogen bonding with Arg-298 from monomer B across the dimer interface. The corresponding residue in monomer B of MERS-CoV 3CLpro and HKU4-CoV 3CLpro is Met-298, which does not participate in any interaction with Thr-126 from monomer A across the dimer interface.
FIGURE 8.
FIGURE 8.
Sequence alignment of 3CLpro enzymes from MERS-CoV, HKU5-CoV, HKU4-CoV, and SARS-CoV. Programs MultAlin (64) and ESPript (65) were used for the sequence alignment and visualization. Secondary structural elements of MERS-CoV 3CLpro are represented as spirals for α-helix, arrows for β-strands, η for 310 helix, and T for β-turns. Residues Val-4 and Met-298 in MERS-CoV, HKU5-CoV, HKU4-CoV 3CLpro, and Arg-4 and Arg-298 in SARS-CoV are shown in a green box; catalytic residues His-41 and Cys-148 are highlighted in a purple box. The nonconserved residues of MERS-CoV 3CLpro are marked with pink arrows. % identity with MERS-CoV 3CLpro is shown.
FIGURE 9.
FIGURE 9.
Analysis of the nonconserved amino acids of MERS-CoV 3CLpro. A, representation of MERS-CoV 3CLpro dimer with monomers A and B colored in orange and yellow, respectively. Nonconserved residues that are present in the loop regions are shown as spheres in gray and pink for monomers A and B, respectively. Other nonconserved residues are represented as spheres with the corresponding chain color. Domains I–III and the inter-domain loop are labeled. Catalytic residues His-41 and Cys-148 are shown as green spheres. Inhibitor molecule is shown in both active sites in blue sticks. B–G, residues of monomer B are shown (yellow and pink), unless otherwise labeled. B, clustering of some of the nonconserved amino acids, His-8, Asp-12, Ala-15, Thr-128, Lys-155, and Ser-158, near the N-terminal region is shown. N-terminal helices for both monomers are labeled. C, His-8 from the N-terminal region forms van der Waals contacts with Lys-155 of the same monomer and Thr-128 of the other monomer in the dimer. D, nonconserved residue Met-61 forms hydrophobic contacts with the Met-43 residue, which is in close proximity to the catalytic residue His-41. E, loop containing the nonconserved residue Ala-171 forms the S1 pocket along with residues His-166 and His-175. F, Val-132 forms hydrophobic contacts with a residue within the same domain (Ala-114), as well as Glu-290 from domain III. G, nonconserved residue Tyr-137 makes hydrophobic contacts with Tyr-185; Tyr-185 along with two other nonconserved residues Thr-183 and Met-189 are present on the inter-domain loop.
FIGURE 10.
FIGURE 10.
Proposed model for polyprotein processing in MERS-CoV regulated by ligand-induced dimerization of MERS-CoV 3CLpro. MERS-CoV 3CLpro domains I and II are together represented as the rectangular box, and domain III is represented as a cylinder. The N and C termini are labeled, and the yellow cylinder labeled S represents a ligand that can be a peptide inhibitor, peptide substrate, or 3CLpro cleavage sites in the polyprotein. Various steps required for the auto-release of 3CLpro from the polyprotein and subsequent processing of the polyprotein cleavage sites are described in the text. Suggested by our AUC and kinetic studies, the shaded region (steps 5 and 6) highlights the additional steps MERS-CoV 3CLpro would undertake during polyprotein processing and have been described in the kinetic model depicted in Fig. 5B.
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References

    1. Alenius S., Niskanen R., Juntti N., Larsson B. (1991) Bovine coronavirus as the causative agent of winter dysentery: serological evidence. Acta Vet. Scand. 32, 163–170 - PMC - PubMed
    1. Barber D. M., Nettleton P. F., Herring J. A. (1985) Disease in a dairy herd associated with the introduction and spread of bovine virus diarrhoea virus. Vet. Rec. 117, 459–464 - PubMed
    1. Perlman S., Netland J. (2009) Coronaviruses post-SARS: update on replication and pathogenesis. Nat. Rev. Microbiol. 7, 439–450 - PMC - PubMed
    1. McIntosh K., Becker W. B., Chanock R. M. (1967) Growth in suckling-mouse brain of “IBV-like” viruses from patients with upper respiratory tract disease. Proc. Natl. Acad. Sci. U.S.A. 58, 2268–2273 - PMC - PubMed
    1. Cavallaro J. J., Monto A. S. (1970) Community-wide outbreak of infection with a 229E-like coronavirus in Tecumseh, Michigan. J. Infect. Dis. 122, 272–279 - PMC - PubMed

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