Abstract
The 3C-like proteinase (3CLpro) 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 3CLpro is active. However, as the internally encoded 3CLpro 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 3CLpro (XX), SARS 3CLpro (3CLP), and the yellow fluorescent protein (Y) to study the autoprocessing of 3CLpro using fluorescence resonance energy transfer. In contrast to the matured 3CLpro, 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-3CLpro in the polyprotein, and a modified model for the 3CLpro maturation process was proposed. As many viral proteases undergo a similar maturation process, this model might be generally applicable.
Highlights
After its outbreak in 2003, severe acute respiratory syndrome (SARS)2 was confirmed to be caused by a new type of coronavirus, SARS-CoV
Substrate-induced Dimerization Is Essential for the Activity of SARS-3C-like proteinase (3CLpro) in the Polyprotein—In vitro studies showed that dimerization is essential for the enzyme activity of SARS3CLpro [4]
Hsu et al [22] reported that the partially cleaved polyprotein can form a small amount of active dimer and proposed that during the autocleavage of the 3CLpro, the enzyme is dimeric at each step
Summary
Plasmid Construction of the Large Fusion Proteins His-CXX(Q/E)-3CLP-Y, His-C-XX-C145A-Y, 3CLP-Y-His, and 3CLPMBP-His—The large fusion protein constructs basically contained four components in sequence. Fusion Protein Expression and Purification—The large polyproteins with His tags were expressed and purified with a slightly modified procedure when compared with the matured enzyme [4] (see detailed information in supplemental material). Enzyme concentration ranges used here were 2.25– 45 M for His-CXX(Q/E)-3CLP-Y, 1.36 –21.78 M for 3CLP-Y-His, and 0.225– 4.5 M for SARS-3CLpro in a 100-l volume, respectively. The enzyme concentration dependence of rate constants was measured at 37 °C in buffer A with 5 mM dithiothreitol. We have tried to fit Equation 1 and found that the activities of monomers were negligible for the matured 3CLpro, as well as the polyproteins. This confirmed again that dimer is the active form. Chemical Cross-linking—Enzymes (6 mg/ml His-C-XX(Q/ E)-3CLP-Y, 6 mg/ml 3CLP-Y-His, and 5.12 mg/ml SARS3CLpro) alone or mixed with equal molar 5f were cross-linked by 10-fold molar excess ethylene glycol bis (succinimidyl succinate) in buffer A at room temperature for 30 min and quenched by adding Tris (1 M, pH 7.5) to a final concentration of 50 mM
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