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Structural and functional characterization of MERS coronavirus papain-like protease

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.


Background
In September 2012, a new highly pathogenic human coronavirus (CoV) 1 , Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries [1][2][3]. The virus causes symptoms similar to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), yet involving an additional component of acute renal failure [4]. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally (World Health Organization, global alert and response, http://www.who.int/csr/don/2014_05_15_mers/en/). Recently, human-to-human transmission of MERS-CoV has been confirmed; albeit, a serological study of major livestock suggested dromedary camels also to be a possible host [5,6]. Nevertheless, these findings indicate that the virus have the opportunity to spread globally and pose a significant threat to world health and the economy. Therefore, studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent.
Like other CoVs, the MERS-CoV nonstructural polyproteins (pp1a and pp1ab) are cleaved by two types of viral cysteine proteases, a main protease (EC 3.4.22.69) and a papain-like protease (PL pro ) (EC 3.4.22.46) [7]. This processing is considered to be a suitable antiviral target because it is required for viral maturation. Unfortunately, initial screening of the existing SARS-CoV PL pro inhibitor, a benzodioxolane derivative against MERS-CoV PL pro , revealed no significant inhibition [7]. The difference represents the requirement of further understanding the MERS-CoV PL pro . In addition to proteolytic activity, similar to those of SARS-CoV, NL63-CoV and murine hepatitis virus, MERS-CoV PL pro acts on both deubiquitination and ISG15-linked ISGylation [8][9][10][11]. As a viral deubiquitinating protease (DUB), MERS-CoV PL pro is able to deubiquitinate interferon regulatory factor 3 (IRF3), which can prevent its nuclear translocation and suppress production of interferon β [10]. These studies support the multifunctional nature of coronaviral PL pro . Recently, with the crystal structure of SARS-CoV PL pro C112S mutant in complex with ubiquitin (Ub), we have demonstrated that Ub core (residue 1-72) makes mostly hydrophilic interactions with PL pro , while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PL pro , mimicking the P4-P1 residues [12]. This bound pattern is similar to that of the ubiquitin-specific proteases (USPs), one of the five distinct DUB families [13,14].
The MERS-CoV PL pro domain in nsp3 of the pp1a proteins (residue 1484-1800) has been identified [7,10,15]. Like other PL pro , there is a catalytic triad consisting of the residues Cys1592, His1759 and Asp1774. Homology modeling suggests that MERS-CoV PL pro , similar to other known PL pro , may have a right-hand-like architecture constituted by palm, thumb, and fingers domains, although their sequence identity are only about 30% [12]. Furthermore, MERS-CoV PL pro is able to recognize and cleave at the LXGG consensus cleavage site, which is essential for most CoV PL pro -mediated processing [10]. Despite this large body of knowledge on MERS-CoV PL pro , in the absence of detailed structural and functional characterization, the molecular basis for its catalytic mechanism remains poorly unknown.
Here, we expressed and purified the MERS-CoV PL pro by E. coli with high yield and high purity. The secondary, tertiary and quaternary structure of MERS-CoV PL pro was then investigated by circular dichroism (CD) spectroscopy, Tyr/Trp fluoresecence and analytical ultracentrifugation (AUC), respectively. The kinetic and DUB activity assays indicated that MERS-CoV PL pro exhibits potent DUB activity but lower proteolytic activity, compared with SARS-CoV PL pro . The present study provides a foundation for understanding the structural and biochemical properties of coronaviral PL pro family, which is applicable to develop strategies inhibiting PL pro for the effective control of highly pathogenic coronaviral infection.

Expression plasmid construction
The sequence of MERS-CoV PL pro (GenBank accession number NC_019843.2; polyprotein residues 1484-1800) was synthesized (MDBio Inc.), digested by NcoI-XhoI and then inserted into the pET-28a(+) vector (Novagen). In the construct, the 6 x His tag was retained at the C-terminus. The reading frame was confirmed by sequencing.

Expression and purification of MERS-CoV PL pro
The expression vector was transformed into E. coli BL21 (DE3) cells (Novagen). For large scaled protein expression, cultures were grown in LB medium of 0.8 liter at 37°C for 4 h, induced with 0.4 mM isopropyl-β-D -thiogalactopyranoside, and incubated overnight at 20°C. After centrifuging at 6,000 x g at 4°C for 15 min, the cell pellets were resuspended in lysis buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 5% glycerol, 0.2% Triton X-100, and 2 mM β-mercaptoethanol) and then lysed by sonication. The crude extract was then centrifuged at 12,000 x g at 4°C for 25 min to remove the insoluble pellet. The supernatant was incubated with 1-ml Ni-NTA beads at 4°C for 1 h and then loaded into an empty column. After allowing the supernatant to flow through, the beads were washed with washing buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 8 mM imidazole, and 2 mM β-mercaptoethanol), and the protein was eluted with elution buffer (20 mM Tris, pH 8.5, 30 mM NaCl, 150 mM imidazole, and 2 mM β-mercaptoethanol). The protein was then loaded onto a S-100 gel-filtration column (GE Healthcare) equilibrated with running buffer (20 mM Tris, pH 8.5, 100 mM NaCl, and 2 mM dithiothreitol). The purity of the fractions collected was analyzed by SDS-PAGE and the protein was concentrated to 30 mg/ml by Amicon Ultra-4 10-kDa centrifugal filter (Millipore).

Circular dichroism spectroscopy
CD spectra of the recombinant MERS-CoV PL pro using a JASCO J-810 spectropolarimeter showed measurements from 250 to 190 nm at 20°C in 50 mM phosphate pH 6.5. The protein concentration was 1.0 mg/ml. In wavelength scanning, the width of the cuvette was 0.1 mm. The far-UV CD spectrum data were analyzed with the CDSSTR program [16,17]. In this analysis, the α-helix, β-sheet, and random coil were split. To estimate the goodness-of-fit, the normalized root mean square deviation was calculated.

Spectrofluorimetric analysis
The fluorescence spectra of the enzyme at 1 μM were monitored in a Perkin-Elmer LS50B luminescence spectrometer at 25°C. The excitation wavelength was set at 280 nm, and the fluorescence emission spectrum was scanned from 300 to 400 nm. Measurement in the maximal peak, intensity, and average emission wavelength were used to confirm the protein folding [18,19].

Analytical ultracentrifugation analysis
The AUC experiments were performed on a XL-A analytical ultracentrifuge (Beckman Coulter) using an An-50 Ti rotor [12,[19][20][21][22]. The sedimentation velocity experiments were performed using a double-sector epon charcoal-filled centerpiece at 20°C with a rotor speed of 42,000 rpm. Protein solutions of MERS-CoV PL pro (1.0 mg/ml) (330 μl) and reference (370 μl) solutions were loaded into the centerpiece, respectively. The absorbance at 280 nm was monitored in a continuous mode with a time interval of 300 s and a step size of 0.003 cm. Multiple scans at different time intervals were then fitted to a continuous c(s) distribution model using the SEDFIT program [23]. All size-and-shape distributions were analyzed at a confidence level of p = 0.95 by maximal entropy regularization and a resolution N of 200 with sedimentation coefficients between 0 and 20 S or molar mass between 0 and 1000 kDa.

Steady-state kinetic analysis
The peptidyl substrate, Dabcyl-FRLKGGAPIKGV-Edans, was used to measure the enzymatic activity of MERS-CoV PL pro and its mutants throughout the course of the study as described [24]. Specifically, the enhanced fluorescence emission upon substrate cleavage was monitored at excitation and emission wavelengths of 329 and 520 nm, respectively, in a PerkinElmer LS 50B luminescence spectrometer. Fluorescence intensity was converted to the amount of hydrolyzed substrate using a standard curve drawn from the fluorescence measurements of well-defined concentrations of Dabcyl-FRLKGG and APIKGV-Edans peptides in a 1:1 ratio. This will also correct for the inner filter effect of the substrate. For the kinetic analysis, the reaction mixture contained 4-50 μM peptide substrate in 50 mM phosphate pH 6.5 in a total volume of 1 mL. After the addition of the enzyme to the reaction mixture, the increase in fluorescence was continuously monitored at 30°C. The increase in fluorescence was linear for at least 3 min, and thus the slope of the line represented the initial velocity (v). The steady-state kinetic parameters of the enzyme were determined by fitting the Michaelis-Menten equation (eq. 1) to the initial velocity data in which k cat is the rate constant, [E] and [S] denote the enzyme and substrate concentration, and K m is the Michaelis-Menten constant for the interaction between the peptide substrate and the enzyme.

Deubiquitination assay
The fluorogenic substrate Ub-7-amino-4-trifluoro-methylcoumarin (Ub-AFC) (Boston Biochem) added at 0.5 or 1.0 μM to 50 mM phosphate pH 6.5 was used for deubiquitination assays as described [12]. The enzymatic activity at 30°C was determined by continuously monitoring the fluorescence emission and excitation wavelength of 350 and 485 nm, respectively.

Results and discussion
Recombinant MERS-CoV PL pro preparation To date, there are still no studies describing the expression and purification of MERS-CoV PL pro proteins. In the present study, the expression vector was constructed and then various E. coli. strains such as BL21 (DE3) STAR (Invitrogen) and Rosetta (DE3) (Novagen) were used to explore heterologous expression of MERS-CoV PL pro . Finally, it was found that the STAR strain showed the best expression efficiency. After expressing the protein in E. coli and purification by nickel affinity chromatography and gel-filtration, the purity of recombinant PL pro was about 99% ( Figure 1A). The size of the recombinant MERS-CoV PL pro was found to be between 30 and 45 kDa, which conforms to the theoretical mass (36.5 kDa). The typical yield was about 42 mg after purification from 0.8 liter of E. coli culture (Table 1). After gel-filtration chromatography, the specific proteolytic activity of PL pro was 4 U/mg, increased by 5-fold, with 49.4% recovery rate. Furthermore, the recombinant MERS-CoV PL pro was digested by trypsin and then analyzed by MALDI mass spectrometry to confirm the amino acid sequence (Additional file 1: Figure S1). The molecular weight of fifteen peptides, which covered 60% amino acid sequence, was observed and confirmed ( Figure 1B and Figure 1C). It indicated that our expression and purification of MERS-CoV PL pro by E. coli is successful. For convenience, in the present studies, the MERS-CoV PL pro domain (polyprotein 1a 1484-1800) is numbered to residue 2 to 317, while the first residue is a methionine.
Secondary, tertiary and quaternary structure analysis of MERS-CoV PL pro Next, secondary, tertiary and quaternary structures of MERS-CoV PL pro were investigated, respectively. CD measurement displayed a spectrum which shows negative ellipticity between 240 and 205 nm and positive between 205 and 190 nm (Figure 2). After analyzed by CDSSTR method [16], the best-fit result showed that MERS-CoV PL pro has 23% of α-helix, 31% of β-sheet, and 46% of random coil. The consist is close to that of SARS-CoV PL pro (pdb code: 4M0W) by X-ray crystallography, which has 26% of α-helix, 36% of β-sheet, and 38% of random coil [12]. It suggests that both PL pro may have a similar scaffold.
The Tyr/Trp fluorescence of MERS-CoV PL pro at the phosphate buffer without or with 9 M urea were also identified ( Figure 3). The measurement indicated that the fluorescent intensity of native PL pro (Figure 3, close circles) shows a 70% increase, as compared with that of the denatured form in urea (Figure 3, open circles). On the other hand, the fluorescence emission spectrum of the native MERS-CoV PL pro shows a maximum at 336 nm, while that of the unfolded one shifts to 340 nm. The tendency is similar to that of SARS-CoV PL pro [19] and suggests a folded structure. Next, we also performed AUC experiments to characterize the   quaternary structure of MERS-CoV PL pro . Figure 4A shows a typical absorbance trace at 280 nm of the PL pro during the experiment. After fitting the signals to a continuous size-distribution model, it was clear that the PL pro was monomeric with a sedimentation coefficient of 2.8 S and molar mass of 35.5 kDa ( Figure 4B and Figure 4C), consistent with that for SARS-CoV PL pro [12,19]. All of these biophysical observation confirmed that the PL pro of MERS-CoV and SARS-CoV should have a very similar structure; albeit they only show 30% sequence identity and 50% similarity [12]. Recent studies hypothesized that the homology model of MERS-CoV PL pro , like other coronaviral PL pro , is a right-handlike architecture consisting of palm, thumb and fingers domains [10,25].

Proteolytic activity of MERS-CoV PL pro
Besides the structural similarity, previous studies have suggested that MERS-CoV PL pro is also a multifunctional enzyme with protease, deubiquitinating and interferon antagonist activities [10]. MERS-CoV PL pro has a catalytic triad which is able to recognize and cleave at LXGG consensus cleavage sites; however, the detail enzyme kinetic mechanism is not known. Here we used the peptidyl substrate, Dabcyl-FRLKGGAPIKGV-Edans, to measure the proteolytic activity of MERS-CoV PL pro ( Figure 5A and Table 2). Interestingly, compared with that  of SARS-CoV, MERS-CoV PL pro is less active, with a 22fold loss in k cat /K m , as a result of a 27.5-fold loss in k cat and 1.3-fold loss in K m . According to the sequence alignment and homology modeling, most important residues for the catalysis, including the catalytic triad, Cys110-His277-Asp292 and the residues for substrate P4-P1 binding, Asp164, Pro249, and Gly276 (Asp165, Pro249, and Gly272 in SARS-CoV PL pro ) are highly conserved ( Figure 6) [10,12]. Previous studies have confirmed that Y265F mutant of SARS-CoV PL pro still maintained comparable proteolytic activity with the wild-type [12]. It indicates that the equivalent residue in MERS-CoV PL pro , Phe268, is able to make a hydrophobic contact with the substrate P4 residue (Ub-Leu73). Furthermore, although different to the residue Tyr269 of SARS-CoV PL pro , the equivalent residue Glu272 of MERS-CoV PL pro , whose carboxyl group can point toward outside the hydrophobic pocket, may not interfere the binding of substrate P4 residue ( Figure 6). By contrast, as a putative oxyanion bound residue (Tyr107 in SARS-CoV PL pro ) [15], the equivalent residue Leu105 of MERS-CoV PL pro cannot provide any hydrogen bonding interaction with oxyanion ( Figure 6). It will disfavor the formation of tetrahedral intermediate. Otherwise, different to the Leu163 of SARS-CoV PL pro , the distinct circular structure of the equivalent residue Pro162 of MERS-CoV PL pro may be too short to hover above the active site for substrate binding and serve to enhance the nucleophilicity of the catalytic triad residue, Cys110 ( Figure 6). These two point mutations in MERS-CoV PL pro may significantly lower the catalytic efficiency.
To verify this, we produced the L105W and P162L mutants of MERS-CoV PL pro , and our kinetic data showed that the L105W mutant has a 23-fold increase in activity measured based on k cat /K m , as a result of a 41-fold increase in k cat and 1.9-fold increase in K m ( Figure 5A and Table 2). The results conform to our prediction. However, in contrast, the P162L mutant has a 67-fold loss in k cat /K m , as a result of a 40-fold loss in k cat and 1.6-fold increase in K m ( Figure 5A and Table 2). It suggests the requirement of the Proline residue in this site, although the reason is still not known. Nevertheless, the significant activity recovery by L105W mutation confirms the essential role of this residue on coronaviral PL pro catalysis. Theoretically, PL pro with lower proteolytic  activity may result in late maturation of viral nsp1, nsp2, and nsp3 proteins; nonetheless, its influence on MERS-CoV remains unknown.

DUB activity of MERS-CoV PL pro
To characterize the DUB activity of MERS-CoV PL pro , the fluorogenic substrate Ub-AFC was used. Interestingly, in contrast with its rather low proteolytic activity, MERS-CoV PL pro shows comparable DUB activity to SARS-CoV PL pro (Table 2 and Figure 5B). It suggests that the two PL pro may show similar binding ability to the Ub core domain (residue 1-72). However, it is inconsistent with our previous observation on the structure of SARS-CoV PL pro in complex with Ub [12]. As mimicking the equivalent residue of MERS-CoV PL pro , the arginine mutation of a key residue for Ub core domain binding, Glu168, can result in unstable binding of SARS-CoV PL pro and Ub and significant loss of DUB activity [12]. To verify this inconsistency, a structure of MERS-CoV PL pro in complex with Ub is quite necessary. Structural characterization of type 1 and type 2 PL pro have revealed that there are four cysteine residues coordinating to a zinc ion within the fingertips region in the finger domain [25,28]. Remove of zinc from SARS-CoV PL pro will cause the tertiary structure more unstable and lead to less active [19]. Based on sequence alignment, MERS-CoV PL pro also has four cysteine residues (Cys190, Cys193, Cys225 and Cys227) on the corresponding position. Here the DUB activity of MERS-CoV PL pro in various EDTA was examined to delineate the possible metal ion effect. The activity was 79% in 10 mM, and 72% left in 50 mM EDTA ( Figure 5B). These results suggest the existence of endogenous metal ion, which is beneficial for its DUB activity. By the way, it has been clarified that exogenous zinc ion can efficiently inhibit SARS-CoV PL pro with the IC 50 value of 1.3 μM [24,29]. Here we also confirmed the potent inhibitory effect of zinc ion on MERS-CoV PL pro ( Figure 5B); whereas the mechanism of this inhibition by zinc is not yet understood.

Conclusions
In summary, following our protocol, active MERS-CoV PL pro can be expressed by E. coli and purified with high yield and high purity. The secondary, tertiary and quaternary structural studies concluded that MERS-CoV PL pro has a similar scaffold to other coronaviral PL pro , as a right-hand-like architecture consisting of palm, thumb and fingers domains. The result of functional assay indicated that MERS-CoV PL pro exhibits potent DUB activity but rather low proteolytic activity. A natural mutation, Leu105, is the major reason for this difference. The present study not only demonstrates the structural and functional characterization of MERS-CoV PL pro , but provides a foundation for further understanding the coronaviral PL pro family, which is an ideal antiviral target. Next, with pure protein and effective proteolytic activity assay, potent inhibitors of MERS-CoV PL pro can be high throughput screened and identified.

Additional file
Additional file 1: Figure S1. Mass spectrometry of trypsin-digested peptides of the recombinant MERS-CoV PLpro protein. The red peaks show the signals of the peptides with correct mass, while the blue ones show the signals of the peptides with oxidation. X-axis indicates the m/z ratio and Y-axis shows the absorbance intensity.