Residues on the dimer interface of SARS coronavirus 3C-like protease: dimer stability characterization and enzyme catalytic activity analysis

Abstract

3C-like protease (3CL pro) plays pivotal roles in the life cycle of severe acute respiratory syndrome coronavirus (SARS-CoV) and only the dimeric protease is proposed as the functional form. Guided by the crystal structure and molecular dynamics simulations, we performed systematic mutation analyses to identify residues critical for 3CL pro dimerization and activity in this study. Seven residues on the dimer interface were selected for evaluating their contributions to dimer stability and catalytic activity by biophysical and biochemical methods. These residues are involved in dimerization through hydrogen bonding and broadly located in the N-terminal finger, the alpha-helix A' of domain I, and the oxyanion loop near the S1 substrate-binding subsite in domain II. We revealed that all seven single mutated proteases still have the dimeric species but the monomer-dimer equilibria of these mutants vary from each other, implying that these residues might contribute differently to the dimer stability. Such a conclusion could be further verified by the results that the proteolytic activities of these mutants also decrease to varying degrees. The present study would help us better understand the dimerization-activity relationship of SARS-CoV 3CL pro and afford potential information for designing anti-viral compounds targeting the dimer interface of the protease.

Fig. 1.

The dimeric structure of SARS-CoV 3CL^pro and extensive residue–residue interactions on the dimer interface. (A) A ribbon diagram for the crystal structure of SARS-CoV 3CL^pro (PDB: 1UK2). Monomer A and B are represented as black and grey, respectively and the three domains are also labelled. The residues involved in monomer–monomer interactions, which were selected for subsequent single point mutation analyses, are shown in the bond model. The binding peptide substrate (MP) is also shown as the stick model. (B) A surface model of the protease. The two monomers are in the same orientation as shown in panel A. (C) The dimer interface between monomer A and B. The bonds and residues belonging to monomer A or B are labelled respectively.

Fig. 2.

Residue–residue interactions. (A) Residue–residue interactions between the N-terminal finger and the S1 subsite in the substrate-binding pocket of SARS-CoV 3CL^pro. (B) Residue–residue interactions between two α–helix A′ (residues 10–15) of domain I in SARS-CoV 3CL^pro dimer. The residues belonging to monomer A or B (PDB: 1UK2) are marked respectively. The labelled residues are shown as sticks, and the rest of the proteins as cartoon. Dashes represent the hydrogen bonds formed on the dimer interface. The hydrophobic interactions between the side-chain phenyl of Phe3 or Phe140 and the neighbouring residues are also labelled as the surface model.

Fig. 3.

CD spectra of the wild type and site-directed mutants for SARS-CoV 3CL^pro. Far-UV CD spectra of the wild type and seven mutated SARS-CoV 3CL^pros at 25°C. Protein concentrations used in CD experiments were 10 μM and all protein samples were prepared in 20 mM sodium phosphate pH 7.5, 100 mM NaCl. The CD spectrum of the wild type protease is shown in black and the spectra of the mutated proteases are shown in light gray.

Fig. 4.

Fluorescence emission spectra of the wild type and site-directed mutants for SARS-CoV 3CL^pro. Fluorescence emission spectra of the wild type and seven mutated proteases were recorded at 25°C after excitation at 280 nm. The protease samples (5 μM) were prepared in 20 mM Tris–HCl pH 7.5, 100 mM NaCl. The spectrum of the wild type protease is shown in black and those of the mutants are shown in light gray.

Fig. 5.

SDS–PAGE profiles of glutaraldehyde cross-linked SARS-CoV 3CL^pros. (A) Cross-linking analysis of the wild type SARS-CoV 3CL^pro. (B–H) Cross-linking analyses of Ser1_Ala, Phe3_Ala, Arg4_Ala, Ser10_Ala, Glu14_Ala, Ser139_Ala and Phe140_Ala mutants, respectively. Lane 1, untreated 3CL^pro (5 mg/ml); lane 2, molecular weight protein standards; lane 3a, 3CL^pro (5 mg/ml) cross-linked by 0.1% glutaraldehyde; lane 3b, 3CL^pro (5 mg/ml) cross-linked by 0.05% glutaraldehyde; lanes 4a and 4b, 3CL^pro (1 mg/ml), 0.1 and 0.05% glutaraldehyde; lanes 5a and 5b, 3CL^pro (0.5 mg/ml), 0.1 and 0.05% glutaraldehyde; lanes 6a and 6b, 3CL^pro (0.2 mg/ml), 0.1 and 0.05% glutaraldehyde.

Fig. 6.

Dimer–monomer equilibria of the wild type and site-directed mutants for SARS-CoV 3CL^pro analysed by SEC. (A) Elution profile of the wild type SARS-CoV 3CL^pro at neutral pH (7.5) and a concentration of 5 mg/ml; (B–(H) Elution profiles of Ser1_Ala, Phe3_Ala, Arg4_Ala, Ser10_Ala, Glu14_Ala, Ser139_Ala and Phe140_Ala mutants at concentrations of 5 mg/ml, respectively. Elution profiles of four marker proteins are also shown in arrow labels. Each protein sample was loaded to a HiLoad 16/60 Superdex 75 prep grade column and then eluted at a flow rate of 1 ml/min with detection of absorbance at 280 nm.

Fig. 7.

Fluorescence profiles of hydrolysis of the fluorogenic substrate by the wild type and site-directed mutants for SARS-CoV 3CL^pro. The fluorogenic substrate at a concentration of 10 μM was incubated with 1 μM wild type or mutated SARS-CoV 3CL^pro in 20 mM Tris–HCl pH7.5, 100 mM NaCl, 5 mM DTT, 1 mM EDTA, at 25°C. Increase of emission fluorescence intensity at 488 nm wavelength was recorded at 10 min intervals, λ_EX = 340 nm. The emission spectrum was recorded for 90 min and the activity of the wild type protease was taken as 100%.