Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase

Abstract

The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays. The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1' positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more beta-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.

Fig. 1

Expression and purification of the SARS 3C-like proteinase. Samples taken at each step of purification were analyzed on a 12% SDS-polyacrylamide gel, and the protein was stained with Coomassie Brilliant Blue. Lane M, protein molecular mass marker; lanes 1 and 2, cell lysates from non-induced and isopropyl-1-thio-β-d-galactopyranoside-induced E. coli BL21(DE3) containing the expression plasmid pET 3CLP-21x; lanes 3 and 4, the supernatant and the deposition separated from the cell lysate by centrifugation; lane 5, collected peak fractions from the nickel-nitrilotriacetic acid column; lane 6, collected peak fractions from the Sephacryl S-200 HR column.

Fig. 2

CD spectra of SARS 3C-like proteinase. The CD spectra of 3C-like proteinase were recorded in 40 mm Tris-HCl buffer, pH 8.0 at 20 °C. a, far-UV CD spectrum shows a positive peak at 196 nm and two negative peaks at 209 and 222 nm, respectively, indicating a mixed α and β structure. b, near-UV CD spectrum shows a broad positive peak at ∼280 nm and a small positive shoulder at 291 nm. c, thermal melting of the protein by monitoring the CD signal at 218 nm gives a sigmoid denaturation curve and a T_m of 61 °C

Fig. 3

Aggregation state of SARS 3C-like proteinase on analytic gel filtration column. Superdex 75 HR column was used on ÄKTA fast protein liquid chromatography for the analysis. Solid line, 4 mg/ml; dot-and-dash line, 0.2 mg/ml. Two peaks corresponding to monomeric and dimeric states were detected at 4 mg/ml, whereas at 0.2 mg/ml, only one peak corresponding to the monomeric state appears.

Fig. 4

Enzyme activity of SARS CoV 3C-like proteinase at different concentrations. The proteolysis activity of expressed SARS CoV 3C-like proteinase at different concentrations was determined by incubating 0.2 mm S01 with 3C-like proteinase in 40 mm Tris-HCl buffer, pH 7.3. The observed k_cat/K_m increases in a linear manner, whereas the enzyme concentration increases, indicating the dimer form is the active form of the proteinase.

Fig. 5

Enzyme activity of SARS CoV 3C-like proteinase at different pH values. The proteolysis activity of expressed SARS CoV 3C-like proteinase at different pH values was determined in citric acid/phosphate buffer (pH 5, 6, 7, and 8) or glycine/NaOH buffer (pH 9 or 10) containing 6.8 mm dithiothreitol, 2 mm S01 as substrate, and 2.14 μm 3C-like proteinase.

Fig. 6

The relationship of secondary structure contents in substrate peptides with cleavage efficiency.a, far-UV CD spectra of peptide substrates. ♦, S01; •, S02; ▴, S05; ▾, S07; and ▪, S11. b, calculated content of secondary structure versus k_cat/K_m profile. This figure shows an interesting tendency of which substrate peptide with small k_cat/K_m has relatively less content of sheet (▪) but more content of unordered structure (○), suggesting that the formation of β-strandlike extended structure is favored for binding to the enzyme and proteolysis.