Maturation Mechanism of Severe Acute Respiratory Syndrome (SARS) Coronavirus 3C-like Proteinase

Abstract

The 3C-like proteinase (3CL pro ) of the severe acute respiratory syndrome (SARS) coronavirus plays a vital role in virus maturation and is proposed to be a key target for drug design against SARS. Various in vitro studies revealed that only the dimer of the matured 3CL pro is active. However, as the internally encoded 3CL pro gets matured from the replicase polyprotein by autolytic cleavage at both the N-terminal and the C-terminal flanking sites, it is unclear whether the polyprotein also needs to dimerize first for its autocleavage reaction. We constructed a large protein containing the cyan fluorescent protein (C), the N-terminal flanking substrate peptide of SARS 3CL pro (XX), SARS 3CL pro (3CLP), and the yellow fluorescent protein (Y) to study the autoprocessing of 3CL pro using fluorescence resonance energy transfer. In contrast to the matured 3CL pro , the polyprotein, as well as the one-step digested product, 3CLP-Y-His, were shown to be monomeric in gel filtration and analytic ultracentrifuge analysis. However, dimers can still be induced and detected when incubating these large proteins with a substrate analog compound in both chemical cross-linking experiments and analytic ultracentrifuge analysis. We also measured enzyme activity under different enzyme concentrations and found a clear tendency of substrate-induced dimer formation. Based on these discoveries, we conclude that substrate-induced dimerization is essential for the activity of SARS-3CL pro in the polyprotein, and a modified model for the 3CL pro maturation process was proposed. As many viral proteases undergo a similar maturation process, this model might be generally applicable.

Structural and Evolutionary Analysis Indicate that the SARS-CoV-2 Mpro is an Inconvenient Target for Small-Molecule Inhibitors Design

“…The MixMD simulations performed with various cosolvents have further confirmed these observations. The largest number and the densest hot-spots were located within the binding cavity and the region essential for Mpros dimerisation [31], between the II and III domains. The deep insight into the local hot-spots distribution of the various cosolvents underlines the large differences in binding sites plasticity.…”

“…A notable number of hot-spots are located around the amino acids that vary between the SARS-CoV-2 and SARS-CoV Mpros (Figure 4, Supplementary Figure S2). The largest number and the densest hot-spots are located within the binding cavity and the region essential for Mpros dimerisation [31], between the II and III domains. The binding cavity is particularly occupied by urea, benzene and phenol hot-spots, which is especially interesting, since these solvents exhibit different chemical properties.…”

Structural basis for the dimerization and substrate recognition specificity of porcine epidemic diarrhea virus 3C-like protease

“…Conversely, dimerization can inhibit an active monomeric enzyme (Marianayagam et al, 2004). In terms of coronavirus 3CL pro s, only the dimeric form is functional (Chen et al, 2008;Li et al, 2010;Shi et al, 2008). In the structure of TGEV 3CL pro , the N-terminus of one monomer helps shape the S1 pocket and the oxyanion hole of the opposite monomer; thus, dimerization is essential for its catalytic activity (Anand et al, 2002).…”

SARS coronavirus main proteinase 3.4.22.69

“…Dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis [41]; <2> SARS-CoV Mpro exists in solution as an equilibrium of both monomeric and dimeric forms, and the dimeric form is the enzymatically active form [40]; <2> wild type and D(300-306) proteases exist with dimer as the major form. The major form becomes monomeric in D(299-306), D(298-306) and D(297-306) [26]) [1,5,16,18,25,26,27,37,40,41] homodimer <2,3> (<2> X-ray crystallography [38]; <2> analytical ultracentrifugation, gel filtration [47]; <3> only the dimeric enzyme is active [51]; <2> tendency of substrate induced dimer formation, gel filtration, analytic ultracentrifugation [50]) [38,47,50,51] monomer <2> (<2> by comparing molecular dynamics simulation of dimer and monomer, the indirect reasons for the inactivation of the monomer are found, that is the conformational variations of the active site in the monomer relative to dimer [25]; <2> the enzyme exists as a mixture of monomer and dimer at a higher protein concentration (4 mg/ml) and exclusively as a monomer at a lower protein concentration [16]; <2> a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ ml. The dimer may be the biological functional form of the protein [27]; <2> in solution the wild type protease exhibits both forms of monomer and dimer and the amount of the monomer is almost equal to that of the dimeric form [37]; <2> wild type and D(300-306) proteases exist with dimer as the major form.…”

“…Mpro-C monomer maintains the same fold as that in the crystal structure of Mpro. On the other hand, the Mpro-C dimer has a novel structure characterized by 3D domain-swapping, which provides the structural basis for the dimer stability) [43] <3> (hanging drop vapor diffusion method, crystals of S139A mutant are grown from the mother liquor containing 0.1 M MES pH 6.0, 10% (w/v) PEG 6000, 1 mM dithiohtreitol, and 5% (v/v) DMSO, crystals of F140A are grown at three pH values in 0.1 M MES pH 6.0/0.1 M MES pH 6.5/0.1 M Tris pH 7.6, with 10% (w/v) PEG 6000, 1 mM dithiothreitol, and 5% (v/v) DMSO) [51] Cloning <1> (mutant enzyme G11A and wild-type enzyme are expressed in Escherichia coli) [19] <2> [6,20,37] <2> (E166A mutant protein expressed in Escherichia coli BL21(DE3)) [47] <2> (His-tagged SARS-CoV 3CL protease expressed in Escherichia coli) [8] <2> (His-tagged artificial polyprotein (cyan fluorescent protein-SARS-CoV 3CLpro-yellow fluorescent protein) expressed in Escherichia coli) [50] <2> (R298A protease is expressed in Escherichia coli strain BL21(DE3)) [41] <2> (expressed in E. coli BL21) [3] <2> (expressed in Escherichia coli) [48,49] <2> (expressed in Escherichia coli BL21 cells) [38] <2> (expression in Escherichia coli) [24,27] <2> (fused to maltose-binding protein and expressed in Escherichia coli BL21) [1] <2> (high level of expression of of proteolysis-resistant mutant R188I in Escherichia coli) [31] <2> (wild type and His-tagged T25G mutant are expressed in Escherichia coli cells) [49] <2> (wild-type and mutant glutathione S-transferase-fusion constructs are transformed into Escherichia coli BL21 cell strain for overexpression) [17] <2> (wild-type enzyme and C-terminally truncated proteases, expression in Escherichia coli) [26] <3> (expressed in Escherichia coli BL21(DE3) cells) [51] Engineering C145A <2> (<2> no irreversible inactivation by benzotriazole esters [13]) [13] C300A <2> (<2> mutant enzyme shows more than 30% of wild-type activity [17]) [17] E14A <2> (<2> the ratio of dimer to monomer in soluti...…”