Structural and functional characterization of MERS coronavirus papain-like protease

Abstract

Backgrounds: A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PLpro) is expressed, and its structural and functional consequences are elucidated.

Results: Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PLpro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PLpro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PLpro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PLpro. A natural mutation, Leu105, is the major reason for this difference.

Conclusions: Overall, MERS-CoV PLpro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro against highly pathogenic coronaviruses.

Figure 1

Expression and purification of recombinant MERS-CoV PL^pro. (A) Protein identification by SDS-PAGE. M: molecular marker. Lane 1–4: cytoplasmic fraction, flow-through, elution from the nickel affinity column and protein fraction from S-100 gel-filtration column. (B) and (C) Protein sequence identification by mass spectrometry. The PL^pro was digested by trypsin and then analyzed by MALDI mass spectrometry. There are 15 matched peptides observed (B) and 60% sequence coverage are shown in bold red (C).

Figure 2

CD spectrum of MERS-CoV PL^pro. The protein of 1 mg/ml was suspended in 50 mM phosphate pH 6.5 and the CD values were measured from 240 to 190 nm at 20°C. The obtained spectrum is shown as close circles and the best fit by CDSSTR [16] is shown by solid line. The normalized root mean square deviation is 0.015.

Figure 3

Fluorescence spectrum of MERS-CoV PL^pro. The protein of 1 μM was dissolved in 50 mM phosphate pH 6.5 (closed circles) or 9 M urea (open circles) and excited with 280 nm UV light. The protein fluorescence emission was monitored from 300 to 400 nm at 25°C.

Figure 4

The continuous size distribution change of MERS-CoV PL^pro. (A) Traces of absorbance at 280 nm of the enzyme in the 50 mM phosphate pH 6.5 during the SV experiment. The protein concentration was 1 mg/ml. For clarity, only every four scan is shown. The symbols represent experimental data and the lines are the results obtained after fitted to the Lamm equation using the SEDFIT program [23,26]. (B) and (C) show the continuous c(s) and c(M) distribution of PL^pro, respectively. The residual bitmap of the raw data and the best-fit results are shown in the inset.

Figure 5

Proteolytic and DUB activity assay of MERS-CoV PL^pro and its mutants. Panel (A) shows the plot of initial velocities versus the concentration of peptidyl substrate, Dabcyl-FRLKGGAPIKGV-Edans. The concentration of the wild-type MERS-CoV PL^pro (by circles), the L105W (by triangles) and P162L mutants (by squares) was 1, 0.1 and 10 μM, respectively. The line represented the best-fit results according to the Michaelis-Menten equation (Eq. 1). The kinetic parameters derived are shown in Table 2. (B) DUB activity analysis. The fluorogenic substrate Ub-AFC (1 μM) was used as the substrate. For comparison, both DUB activity of SARS-CoV and MERS-CoV PL^pro was tested. The protein concentration was 0.17 μM. Besides, the inhibition of MERS-CoV PL^pro by 10–50 mM EDTA or 50 μM Zn²⁺ were also clarified.

Figure 6

Putative active site of MERS-CoV PL^pro. The model structure of MERS-CoV PL^pro (in cyan) was generated by SWISS-MODEL [27] and then overlaid with the structure of SARS-CoV PL^pro (in grey) in complex with Ub (in yellow) (PDB code: 4M0W). The residues are shown as sticks and hydrogen bonding and ion-pair interactions are indicated by red dashed lines. Four residues of SARS-CoV PL^pro, Trp107, Leu163, Tyr265 and Tyr269, are labeled in black. The figure was produced using PyMol (http://www.pymol.org).