pH-dependent conformational flexibility of the SARS-CoV main proteinase (M(pro)) dimer: molecular dynamics simulations and multiple X-ray structure analyses

Abstract

The SARS coronavirus main proteinase (M(pro)) is a key enzyme in the processing of the viral polyproteins and thus an attractive target for the discovery of drugs directed against SARS. The enzyme has been shown by X-ray crystallography to undergo significant pH-dependent conformational changes. Here, we assess the conformational flexibility of the M(pro) by analysis of multiple crystal structures (including two new crystal forms) and by molecular dynamics (MD) calculations. The MD simulations take into account the different protonation states of two histidine residues in the substrate-binding site and explain the pH-activity profile of the enzyme. The low enzymatic activity of the M(pro) monomer and the need for dimerization are also discussed.

Figure 1

Structure of the (a) monomer and (b) dimer of SARS-CoV M^pro. (a) Domains I (light blue) and II (green) each contain a six-stranded β-barrel and domain III (orange) is composed mainly of α-helices. The amino and the carboxy terminus are marked by a blue and an orange sphere, respectively. The flexible loops L1, L2, and L3 (red) comprise residues 138–145 (the oxyanion-binding loop), 165–172, and 185–200, respectively. (b) α-Helices are red and β-strands are light blue. The amino and the carboxy termini are marked by blue and orange spheres, respectively. Dimerization is mainly due to interactions between the helical domains III of each monomer (top). (c) Superimposition (in stereo) of the C^α backbone as determined in three different crystal forms. Blue, monoclinic form; red, tetragonal form; green, orthorhombic form. (a) and (b) were prepared by MOLSCRIPT, (c) was prepared by PyMOL.

Figure 2

Time dependence of the RMSDs from the starting structure of the SARS-CoV M^pro dimer for C^α atoms during the 10 ns MD simulation. (a), (b), (c), and (d) correspond to pH 6.0, pH 7.6, pH 8.0, and pH 5.0, respectively, shown as 10 ps averages.

Figure 3

Residue fluctuations for the SARS-CoV M^pro. (a) and (b) Atomic fluctuations of (a) chain A and (b) chain B over the 10 ns equilibrium simulation at pH 6.0. (c) and (d) Mean atomic deviations (〈r〉 values) computed from the experimentally derived B factors using the equation $〈\Delta r_i^2〉=3B_i/(8{\mathrm{\pi }}^2)$, for (c) chain A and (d) chain B in the SARS-CoV M^pro monoclinic crystal structure (1UJ1.pdb); the corresponding values derived from the new crystal structures are shown in (d) for the tetragonal form (red) and the orthorhombic form (green). (e) and (f) RMSD for (e) monomer A and (f) monomer B for five independent monoclinic crystal structures of SARS-CoV M^pro at pH 6.0, fitted to 1UJ1.pdb, and colored black, yellow, cyan, blue and magenta, respectively. The values for the new crystal structures are shown in (f) for the tetragonal form (red) and the orthorhombic form (green).

Figure 4

Conformations of the catalytic site at pH 6.0. (a) and (b) Monoclinic X-ray structure, protomers A and B, respectively; (c) and (d) 10 ns snapshot of the MD simulation; protomers A and B, respectively. Water molecules are indicated by W. Some key distances (not necessarily hydrogen bonds) are indicated by broken lines and values given in Å. Note the salt-bridge between Asp187 and Arg40 (see the text).

Figure 5

pH-activity curve for SARS-CoV M^pro with a pentadecapeptide substrate (see the text for details).

Figure 6

The S1 binding pocket and the oxyanion loop (residues 138–145, first two residues not shown) as revealed by X-ray crystallography (Yang et al. and this work, monoclinic crystal form). Left panel: monomer A; right panel: monomer B. Residues of the parent monomer are shown in light blue, with the exception of Glu166, which is red. The N-terminal residues of the other monomer in the dimer are shown in dark blue. (a), (b) and (c) The crystal structures at pH 6.0, pH 7.6 and pH 8.0, respectively. To visualize substrate binding to the enzyme, an additional panel is included in (a) (far right) showing the P1 Gln residue (green) of a substrate-analogous inhibitor as bound in the S1 specificity pocket. Note the interaction with His163 at the distal end of the P1 glutamine.

Figure 7

The S1-binding pocket and the oxyanion loop (residues 138–145, first two residues not shown) in the MD simulations (snapshots after 10 ns). (a), (b), (c) and (d) pH 6.0, pH 7.6, pH 8.0 and pH 5.0, respectively. Note that interaction of Glu166 (red) with His172 (to the left) constitutes the active conformation of the SARS-CoV M^pro, whereas its interaction with His163 (to the right) blocks the S1 pocket (compare also Figure 6(a), middle panel) and therefore leads to inactivation of the enzyme.

Figure 8

Some characteristic distances in the MD simulations of the SARS-CoV M^pro dimer at (a) pH 6.0, (b) pH 7.6, (c) pH 8.0, and (d) pH 5.0. For each simulation, the distance between Glu166 and His163, Glu166 and His172, Glu166 and Ser1(N) of the other monomer in the dimer, and Phe140 (center of mass of phenyl ring) and His163 (center of mass of imidazole ring) are shown. The shorter of the two distances to the carboxylate oxygen atoms of Glu166, O^ε1 and O^ε2, is displayed. Green, monomer A; blue, monomer B.

Figure 9

Volume of the binding pocket of protomers A and B at pH 6.0 during the simulation time.