Correlation between dissociation and catalysis of SARS-CoV main protease

Abstract

The dimeric interface of severe acute respiratory syndrome coronavirus main protease is a potential target for the anti-SARS drug development. We have generated C-terminal truncated mutants by serial truncations. The quaternary structure of the enzyme was analyzed using both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. Global analysis of the combined results showed that truncation of C-terminus from 306 to 300 had no appreciable effect on the quaternary structure, and the enzyme remained catalytically active. However, further deletion of Gln-299 or Arg-298 drastically decreased the enzyme activity to 1-2% of wild type (WT), and the major form was a monomeric one. Detailed analysis of the point mutants of these two amino acid residues and their nearby hydrogen bond partner Ser-123 and Ser-139 revealed a strong correlation between the enzyme activity loss and dimer dissociation.

Fig. 1

Structural features of the dimeric SARS-CoV main protease. (A) The enzyme structure (PDB code: 1z1j) is presented as a hollow surface model for protomer A and a ribbon model for protomer B. The catalytic dyad His-41 and Cys-145 (alanine in 1z1j) are shown with stick model in magenta in protomer A. The blue surface region represents the chymotrypsin-like fold. The red surface region indicates the helical domain III. The truncated C-terminal region is also shown with stick in protomer B for residues 300–306 in red, 299 in green, and 298 in blue. Figure generated with MacPyMol . (B) LigPlot of the interfacial region showing the hydrogen bonding of Arg-298 and Gln-299 in protomer B with Ser-123 and Ser-139 in protomer A. The subunit origin is enclosed in parentheses. The symbol keys are shown under the plot.

Fig. 2

CD and fluorescence spectra of the full-length WT and truncated SARS-CoV main proteases. Far-UV CD and fluorescence emission spectra of all recombinant proteases were monitored at 0.5 mg/ml and 8 μg/ml, respectively, in 10 mM PBS buffer (pH 7.6) at 25 °C.

Fig. 3

Continuous sedimentation coefficient distribution of the recombinant SARS-CoV main protease. All enzyme preparations used a concentration of 1 mg/ml in 10 mM PBS buffer (pH 7.6). (A) WT; (B) R298A; (C) Q299A; (D) R298A/Q299A; (E) Δ(300–306); (F) Δ(299–306); (G) Δ(298–306); (H) Δ(297–306). The peaks corresponding to monomer and dimer are indicated.

Fig. 4

Global analysis of the sedimentation velocity and sedimentation equilibrium data of WT SARS-CoV main protease. (A–C) Sedimentation velocity at three protein concentrations: 1.0, 0.2, and 0.04 mg/ml, respectively. (D) Sedimentation equilibrium of 0.2 mg/ml enzyme at three rotor speeds: 8000; 10,000; and 12,000 rpm, each for 12 h. The sedimentation profiles were monitored with the absorbance optical system at wavelength of 280 nm. All spectral data were globally fitted to a monomer–dimer equilibrium model with Lamm equation implemented in the software SEDPHAT . The symbols are the raw sedimentation data and the lines are the theoretical fitted data. The fitting residuals are under each panel.

Fig. 5

Correlation between the enzyme activity loss and subunit dissociation of SARS-CoV main protease. Double logarithmic plot showing the correlation between kinetic parameters and equilibrium dissociation constant (A) or dissociation rate constant (B).