Critical assessment of important regions in the subunit association and catalytic action of the severe acute respiratory syndrome coronavirus main protease

Abstract

The severe acute respiratory syndrome (SARS) coronavirus (CoV) main protease represents an attractive target for the development of novel anti-SARS agents. The tertiary structure of the protease consists of two distinct folds. One is the N-terminal chymotrypsin-like fold that consists of two structural domains and constitutes the catalytic machinery; the other is the C-terminal helical domain, which has an unclear function and is not found in other RNA virus main proteases. To understand the functional roles of the two structural parts of the SARS-CoV main protease, we generated the full-length of this enzyme as well as several terminally truncated forms, different from each other only by the number of amino acid residues at the C- or N-terminal regions. The quaternary structure and K(d) value of the protease were analyzed by analytical ultracentrifugation. The results showed that the N-terminal 1-3 amino acid-truncated protease maintains 76% of enzyme activity and that the major form is a dimer, as in the wild type. However, the amino acids 1-4-truncated protease showed the major form to be a monomer and had little enzyme activity. As a result, the fourth amino acid seemed to have a powerful effect on the quaternary structure and activity of this protease. The last C-terminal helically truncated protease also exhibited a greater tendency to form monomer and showed little activity. We concluded that both the C- and the N-terminal regions influence the dimerization and enzyme activity of the SARS-CoV main protease.

Fig. 1

Structural features of the dimeric SARS-CoV main protease.A, secondary structure of the enzyme and the deleted regions of the various deletion mutants. B, the enzyme structure (Protein Data Bank code 1uk3) is presented as ribbons and labeled as to coincide with the stick model. Protomer A is colored with the catalytic domains I and II in blue, and the helical domain III in purple. The N-finger residues and the last α-helix of protomer A are shown in stick model form in blue and CPK (oxygen in red, nitrogen in blue, sulfur in yellow, and gray for all other atoms), respectively. The catalytic dyad His-41 and Cys-145 are in red. The surface of protomer B is shown in mesh model form. This figure was generated with Spock software. C, the backbone of the enzyme is shown as a stick model in blue and green, and the major pockets are shown as a space-filling model. This figure was generated with CASTp (39).

Fig. 2

CD spectra of the full-length WT and truncated SARS-CoV main proteases. Far-UV CD spectra of all recombinant SARS-CoV main proteases were monitored at a 0.8 mg/ml concentration in 10 mm PBS buffer (pH 7.6) at 25 °C, where deg is the ellipticity in degrees.

Fig. 3

Fluorescence spectra of the full-length WT and truncated SARS-CoV main proteases. Fluorescence emission spectra of all recombinant proteases were monitored at 8 μg/ml concentration in 10 mm PBS buffer (pH 7.6) at 25 °C.

Fig. 4

Continuous sedimentation coefficient distribution of the full-length WT and N-terminally truncated SARS-CoV main protease. The residual bitmaps of various main proteases are shown in the insets. All enzyme preparations used a concentration of 1 mg/ml in 10 mm PBS buffer (pH 7.6) at 20 °C. A, WT; B, Δ(1–3); C, Δ(1–4); D, Δ(1–5); E, Δ(1–6); F, Δ(1–7). The left dotted line indicates the monomer, and the right one is the dimer form.

Fig. 5

Continuous sedimentation coefficient distribution of the full-length WT and C-terminally truncated SARS-CoV main protease. The residual bitmaps of various main proteases are shown in the insets. All enzyme preparations used a concentration of 1 mg/ml in 10 mm PBS buffer (pH 7.6) at 20 °C. A, WT; B, Δ(293–306); C, Δ(201–306). The left dotted line indicates the monomer, and the right one is the dimer form.

Fig. 6

Global analysis of the sedimentation velocity data of full-length WT SARS-CoV main protease at three protein concentrations. Sedimentation was performed at 20 °C with an An50 rotor and at rotor speeds of 42,000 revolutions/min. A–C, the concentrations of the protein were 0.1 mg/ml, 0.5 mg/ml, and 1 mg/ml, respectively. The sedimentation profiles are from the absorbance optical system at a wavelength of 280 nm. The symbols are the raw sedimentation data, and the lines are the theoretical fitted data to the Lamm equation implemented in the software SEDPHAT. D–F, the randomly distributed residuals of the fitting model from the upper panel provide a credible analysis result for a dissociation constant (K_d) of the monomer-dimer equilibrium.

Fig. 7

Interfacial regions of the dimeric SARS-CoV main protease. The interfacial contact surface regions within 1 nm for both protomers A and B are shown in mesh model form. The same surface areas were turned 90° around the x-axis to give C and D, respectively, from A and B. The C-terminal helical region (red) and the N-terminal finger (blue) are shown in stick mode. This figure was generated with Spock software.