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Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan
Correspondence: Address reprint requests to Hui-Ping Chang, Dept. of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, 155 Li-Nong St., Section 2, Taipei 112, Taiwan. E-mail: huiping_chp{at}hotmail.com.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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). A specific SARS coronavirus (CoV) was identified to be the contagious agent. Maturation of the SARS-CoV involves proteolytic processing of the virus polyproteins by the chymotrypsin-like main protease (3CLpro) and a papain-like protease (PLpro). SARS-CoV might invade cells by a protease-mediated endosomal pathway (2). Any knowledge about the structural stability of viral protease can be very rewarding for the development of antivirus drugs (3–6).
The 3CLpro from SARS-CoV was the first protein of this virus to have its three-dimensional structure solved (7,8). As had previously been shown for the 3CLpro of transmissible gastroenteritis (corona)virus (9), the SARS-CoV enzyme also exists as a dimer in solution and in crystals, with each subunit containing three structural domains (Fig. 1). The catalytic dyad His41-Cys145 is located between the interfaces of domains I (residues 8–99) and domain II (residues 100–183), which constitute the chymotrypsin-like folding scaffold. This catalytic N-terminal domain is connected by a long loop (residues 184–199) to the helical domain III (residues 200–306). The functional role of the extra C-terminal domain III is less clear. Numerous experimental evidence shows the importance of N- and C-terminal residues in the dimerization (4,10–12). Structural studies indicate the close proximity of C-terminus and N-finger (N-terminal residues 1–7) (9) from subunit A to the active site of subunit B that led to the development of a novel autocleavage mechanism that can explain the dimeric nature of the mature enzyme (13).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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Site-directed mutagenesis (14) was used to construct pET-H41A, pET-S65A, pET-N84A, pET-C145A, pET-Y161A, and pET-H163A vectors. The forward primers for H41A, S65A, N84A, C145A, Y161A, and H163A were 5'-CTGTCCAAGAGCGGTCATTTGCAC-3', 5'-CAAATCCAACCATGCGTTTCTTGTTC-3', 5'-CATTCTATGCAAGCGTGTCTGCTTAG-3', 5'-CTTAATGGATCAGCGGGTAGTGTTG-3', 5'-GTCTTTCTGCGCGATGCATCATATG-3', and 5'-CTGCTATATGGCGCATATGGAGC-3', respectively; and the reverse primers were 5'-GTGCAAATGACCGCTCTTGGACAG-3', 5'-GAACAAGAAACGCATGGTTGGATTTG-3', 5'-CTAAGCAGACACGCTTGCATAGAATG-3', 5'-CAACACTACCCGCTGATCCATTAAG-3', 5'-CATATGATGCATCGCGCAGAAAGAC-3', and 5'-GCTCCATATGCGCCATATAGCAG-3', respectively. The pET-3CL expression vectors were used as templates and the primers as described above were used to mutate the His41, Ser65, Asn84, Cys145, Tyr161, and His163 codons to alanine codon by PCR. The mutational sites are shown underlined and in bold.
For the tryptophan mutants of 3CLpro, the forward primers for W31F, W207F, and W218F were 5'-CTCTTAATGGATTGTTCTTGGATGACACAGT-3', 5'-CCATAACATTAAATGTTTTGGCATTCCTGTATGCTGC-3', and 5'-CAATGGTGATAGGTTCTTTCTTAATAGATTCACC-3', respectively. The reverse primers were 5'-ACTGTGTCATCCAAGAACAATCCATTAAGAG-3', 5'-GCAGCATACAGGAATGCCAAAACATTTAATGTTATGG-3', and 5'-GGTGAATCTATTAAAGAAGAACCTATCACCATTG-3', respectively. For W207F/W218F (W31) double mutant, W207F was used as the template.
All nucleotide sequences were confirmed by autosequencing analysis. A sequence comparison bearing the mutation positions at the active site region is shown in supplemental Fig. 1S (Supplementary Material). The wild-type (WT) enzyme and all mutants were successfully expressed and purified to apparent homogeneity by protocols described previously (11). Typical CD spectra of the recombinant 3CLpro are shown in Supplementary Material (Fig. 2S).
Enzyme activity assay of SARS-CoV main protease using fluorogenic substrate
The enzyme kinetic measurements were performed in 30 mM Tris-Cl buffer (pH 7.7) or phosphate-buffered saline (pH 7.6) at 30°C. Enhanced fluorescence due to cleavage of the internal quenched fluorogenic substrate peptide (ortho-aminobenzoic acid-TSAVLQSGFRK-2,4-dinitrophenyl amide) was monitored at 420 nm with excitation at 362 nm using a Perkin-Elmer LS-50B luminescence spectrometer.
The kinetic parameters were obtained by assaying the enzyme activity at various peptide substrate concentrations around its Michaelis constant (Km) (11). The initial velocities (vo) were measured and fitted to the Michaelis-Menten equation (Eq. 1):
 | (1) |
Reversible unfolding/refolding of the enzyme in GdnCl
WT (3CLpro) or domain III-deleted (3CLI+II) SARS-CoV main protease was unfolded with different concentrations of GdnCl in Tris-Cl buffer (30 mM, pH 7.7) at 30°C for 10 min. The unfolding of the enzyme was monitored by fluorescence, CD, sedimentation velocity, and enzyme activity loss. The unfolding was found to reach equilibrium in transient. A 10-min incubation, both unfolding and refolding, was used in the experiments in this study.
Spectrofluorimetric analysis
Fluorescence spectra of the enzyme were monitored in a Perkin-Elmer LS-50B luminescence spectrometer at 30°C, and all spectra were corrected for buffer absorption. The excitation wavelength was set at 280 nm (295 nm for tryptophan mutants, W31 and W207/W218), and the fluorescence emission spectrum was scanned from 300 to 400 nm. The maximal peak of the fluorescence spectrum and the change in fluorescence intensity were used in monitoring the unfolding processes of the enzyme. Both the red shift and the changes in fluorescence intensity were analyzed together using the average emission wavelength (AEW) (
) according to Eq. 2 (15):
 | (2) |
i). Exposure of the enzyme hydrophobic areas was examined by mixing the enzyme solution with 1-anilino-8-naphthalene sulfonic acid (ANS) and the fluorescence emission spectrum between 400 and 600 nm was recorded with excitation at 395 nm.
Circular dichroism analysis
CD spectra were measured at 30°C with a Jasco J-810 spectropolarimeter under constant N2 flush. The enzyme solution was scanned from 300 to 190 nm. When the analysis was completed, the buffer was immediately scanned to obtain the baseline. Unfolded enzyme solution was filtered through a 0.22-mm filter (Whatman, UK) before analysis. Denaturing agent included in the buffer was used to monitor the baseline for the unfolded enzyme.
Mean residue ellipticity [
] was calculated according to Eq. 3:
 | (3) |
, the observed ellipticity.
Analytical ultracentrifugation
Sedimentation velocity was performed in a Beckman-Coulter XL-A analytical ultracentrifuge with an An50Ti rotor at 20°C and 42,000 rpm in 12-mm double-sector Epon charcoal-filled centerpieces. The ultraviolet (UV) absorption of the cells at 280 nm was scanned in a continuous mode with time interval of 8 min and a step size of 0.003 cm. The partial specific volume of the enzyme, solvent density, and viscosity were calculated by the free software SEDNTERP (http://www.jphilo.mailway.com/). All samples were visually checked for clarity after ultracentrifugation to make sure that there was no indication of precipitation due to unfolding of the protein. Multiple scans at different time points were analyzed with the SEDFIT program (16,17).
The size-and-shape distributions of the enzyme in the presence of GdnCl were analyzed with a new feature of SEDFIT (17) that allowed full exploration of the protein conformational changes. The sedimentation velocity data were fitted using a two-dimensional distribution with respect to frictional ratio c(s, f/fo) according to Lamm equation (17,18) (Eq. 4):
 | (4) |
(s,D,r,t), the solution of the Lamm equation; and D(s, f/fo), the dependence of diffusion coefficient (D) on sedimentation coefficient (s) and frictional ratio (f/fo), where
 | (5) |
 | (6) |
denoting angular velocity; k, Boltzmann constant; T, absolute temperature; 
, enzyme partial specific volume; 
, buffer viscosity; and 
, buffer density. All two-dimensional distributions were solved and normalized to a confidence level of p = 0.95 by maximum entropy and a resolution N of 200 with sedimentation coefficients between 0.1 and 20 S. The anhydrous friction ratio is from 1.0 to 2.0 or 3.5 at a resolution of 10.
Unfolding data analysis
Because unfolding/refolding is a reversible process (see Fig. 2), the unfolding data were treated with the following thermodynamic models by global fitting of the data to Eq. 7 or Eq. 8:
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):
 | (Scheme I) |
 | (7) |
The three-state unfolding model (Scheme II) was described by Eq. 8 (20):
 | (Scheme II) |
 | (8) |
GN-I and GI-U are the free energy changes for the N
I and IU processes, respectively; mN-I and mI-U are the sensitivities of the respective unfolding process to denaturant concentration. |
RESULTS |
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). In a recent article, Chen et al. (21) confirmed that the dimeric SARS-CoV main protease is asymmetric and only one protomer is active at a time. They proposed a novel association-activation-catalysis-dissociation mechanism for the enzyme activity control. We checked whether dissociation is the major reason for enzyme inactivation. The following residues were selected for analysis: His41 and Cys145, the catalytic dyad; His163, the substrate-binding residue; Tyr161, residue for holding His163 in place. Some surface residues at domain I, Ser65 and Asn84, were also selected as control. In the Ramachandran plot (22), Asn84 is in the disallowed region of the unbound 3CLpro but in a generously allowed region of a 3CLpro-azapeptide inhibitor complex (23). Table 1 summarizes the kinetic parameters of these mutants. Mutation at catalytic dyad residues (H41A and C145A) abolishes almost the complete biological function while the enzyme exists exclusively as dimer. Assuming that the rate-limiting step of the hydrolytic activity of 3CLpro, like chymotrypsin, is at the covalent acylation-deacylation step, then the Km value for the substrate reflects an apparent binding affinity for the enzyme-substrate complex. However, mutation at S1 site (H163A) of the substrate-binding pocket did not change the substrate binding appreciably as anticipated. The major effect was on the kcat value. H163A possessed 
0.1% of enzyme activity compared with WT. In contrast, the catalytic dyad mutants, H41A and C145A, had only 10–6 relative activity left.
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Reversible unfolding of SARS-CoV main protease in GdnCl
SARS-CoV 3CLpro was denatured at high temperature. The thermal denaturation was irreversible. White coagulations occurred in the cuvettes when the enzyme at 0.3–0.4 mg/ml in phosphate-buffered saline (pH 7.6) was heated to 60°C for 30 min (data not shown). This result was also observed in other studies (24,25). Thermal unfolding is thus not suitable for further analysis of the unfolding process of 3CLpro.
The fluorescence emission spectrum of the native enzyme shows a maximum at 328 nm (Fig. 2 A, curve a). The enzyme was very sensitive to GdnCl. In the presence of 6 M of GdnCl, the enzyme was completely unfolded in 10 min and the maximum emission fluorescence was shifted to 358 nm (Fig. 2 A, curve c). The unfolding was found to be completely reversible. A simple 10-fold dilution instantaneously induced refolding of the enzyme to a yield of 92–95% as judged by the fluorescence spectrum recovery (Fig. 2 A, curve d).
Similar results were obtained for the C-terminal domain-III-truncated enzyme (3CLI+II) in which only one buried tryptophanyl residue remained and thus very little fluorescence was detected in the folded state (Fig. 2 B, curve a). The GdnCl-induced unfolding is thus very suitable for conformational stability studies of the enzyme.
Unfolding curves of SARS-CoV main protease in various concentrations of GdnCl
Unfolding of the enzyme in different concentrations of GdnCl is shown in Fig. 3. When the integrated fluorescence area was calculated at various GdnCl concentrations, a monophasic unfolding process seems sufficient for describing the data (Fig. 3 C). However, there is a strong denaturant-dependent pretransition slope, which might indicate exposure of hydrophobic areas (26). Similar results were obtained with CD spectra (Fig. 3 D). More interesting results are shown in Fig. 3 B, which shows clearly a biphasic red shifting of the fluorescence emission peak from 328 to 358 nm upon addition of GdnCl. The average emission wavelength (Fig. 3 A) that registered changes in both fluorescence wavelength and fluorescence intensity was used to calculate the thermodynamic parameters of the unfolding/refolding process (Table 2).
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0.4 M GdnCl and reached an unfolding intermediate with [GdnCl]0.5,N-I at 1.1 M. The second unfolding phase started at 3 M denaturant with [GdnCl]0.5,I-U = 4.0 M, corresponding to free energy change (G) of 1.22 ± 0.16 kcal/mol, and 10.88 ± 0.27 kcal/mol, respectively, for the N I and I U processes.
Quaternary structural changes of SARS-CoV main protease in the presence of GdnCl
The above results indicate clearly a biphasic unfolding curve for the dimeric enzyme. It is ultimately important to differentiate between dissociation versus unfolding under GdnCl. Analytical ultracentrifugation was used to examine this point. Fig. 4 A shows a typical sedimentation velocity profile for the enzyme in the presence of 0.4 M GdnCl. As shown in Fig. 4, B and C, the enzyme already dissociated to monomers at 0.4 M GdnCl. The major species of 4.6 ± 0.2 S corresponds to a dimer in the absence of GdnCl (Fig. 4 C), whereas in 0.4 M GdnCl the major peak corresponds to a monomer with a value of 2.9 ± 0.2 S. An equivalent continuous distribution C(s,M) plot (not shown) indicated the dissociation of dimeric 3CLpro with molecular mass of 61 ± 17 kDa in buffer to a monomer with molecular mass of 33 ± 6 kDa in 0.4 M GdnCl. The biphasic-unfolding phenomenon is thus not a dissociation-unfolding process. Fig. 3, A and B, then represents a folding/unfolding process of the dissociated monomer.
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The above observed unfolding intermediate may represent a molten globule state with exposure of the interior hydrophobic area, which will have pronounced binding ability to ANS and have a higher propensity to aggregate (27). However, no precipitation was observed for 3CLpro at any of the GdnCl concentrations. Neither was polymerization detected by AUC. ANS binding ability of the GdnCl-unfolded enzyme remained unchanged (Supplementary Material, Fig. 4S). This probably excludes a gross conformational change of the enzyme and the major intermediate form detected at 1–4 M GdnCl may not represent a partially unfolded molten globule state. The remaining possibility is then differential domain stability. The unfolding of 3CLI+II was then examined.
Unfolding curves of the C-terminal domain-truncated SARS-CoV main protease (3CLI+II)
In the presence of GdnCl, 3CLI+II follows the simple monophasic-unfolding curve (Fig. 6), which is also completely reversible (Fig. 2 B). Compared with the unfolding curve of the full-length 3CLpro, it is very likely that the biphasic unfolding of 3CLpro represents differential conformational stability of domain (I + II) and domain III.
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Unfolding curves of the tryptophanyl residue(s)-mutated SARS-CoV main protease
3CLpro has a total of three tryptophanyl residues (Trp31 at domain I, Trp207 and Trp218 at domain III, domain II has no Trp) (Fig. 1 A). W31, a double mutant with both Trp207 and Trp218 of domain III mutated to phenylalanine and only one tryptophanyl residue (Trp31) remained. When excited with 295 nm UV light, the protein intrinsic emission fluorescence came from the tryptophanyl residue only. Thus W31 will only report the structural information of the N-terminal domain. Similarly, W207/W218, a single W31F mutant, will register the conformational change of the C-terminal domain.
Mutation at these tryptophanyl residues to phenylalanine did not change the kinetic properties of the enzyme. The kcat and Km values of W31 or W207/W218 were in the same order as in WT. The AUC data indicated that these Trp mutants still existed as dimer in solution (Table 1). When W31 and W207/W218 were unfolded in GdnCl, the fluorescence signal showed monophasic in both cases but distinct unfolding curves of these mutants were obtained (Fig. 7). It is clear that the N-terminal domain of 3CLpro (W31) is unfolded ([GdnCl]0.5 1 M) before the C-terminal domain (W207/W218) ([GdnCl]0.5 4 M). The unfolding parameters of 3CLI+II thus represented the first phase of the 3CLpro (Table 2).
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DISCUSSION |
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); His41, Cys145, Tyr161 in feline infectious peritonitis virus (28); and Cys145 in SARS-CoV (10,13,29). Table 1 summarizes the results obtained in this study. As expected, the almost complete loss of enzymatic activity of H41A and C145A confirmed the catalytic roles of the catalytic dyad (29,30). Similar observations were made for His163 and Tyr161. Both of these residues are involved in substrate binding. The main function of Tyr161 is to hold His163 in place. Mutation of His163 led to a relatively small increase in Km but a large drop in kcat (Table 1). This could result from the enzyme activity assays being performed at pH 7.6, where one protomer of the dimeric enzyme adopts a different conformation from that in acidic environment (7). It has been postulated by Tan et al. (30) and also by Yang et al. (7) that at low pH, protonation of His163 induces a series of conformational changes that lead to the collapse of the oxyanion hole and blocks the S1 pocket entry. This would be in full agreement with the observation of a very large decrease in kcat as shown in Table 1. An intriguing question will then be testing the pH-activity profile of H163A mutant to confirm that His163 is responsible for the lower activity of 3CLpro at pH 6.0 as predicted by molecular dynamic simulation (30).
The contribution of this study includes our presentation of a clear-cut dissociation-denaturation profile as well as a differential domain unfolding process. Protein folding constitutes one of the most challenging problems in the life science area (31–35). The SARS-CoV main protease has a unique structure with distinct structural domains. Each subunit of the protein has 13 strands and 11 helices, distributed in different structural domains (7). A multiphasic unfolding process may be anticipated. From the present analyses, the differential stability of different structural domains of the enzyme was deduced in terms of their free energy changes during transformation from the folded state to the unfolded state with an intermediate state involvement of folded domain III but unfolded domain (I+II) by the following reasoning.
Bacha et al. (24) had demonstrated the intrinsic instability of the C-terminal domain III by differential scanning calorimetry albeit this technique seemingly does not provide enough resolution to detect the unfolding intermediate. We observed a clear biphasic unfolding curve for the 3CLpro by the protein intrinsic fluorescence. The intermediate form was assigned to a folded C-terminal domain but an unfolded N-terminal domain on the basis of the following observations: a), Our unfolding data of 3CLpro clearly demonstrate the biphasic-unfolding process and our data of 3CLI+II (N-terminal domain) suggest attributing the first and second phases of the 3CLpro to the N- and C-terminal domains, respectively (Table 2). b), Unequivocal assignment of the biphasic unfolding curve to differential domain stability came from results shown in Fig. 7. The W31 and W207/W218 are full-length 3CLpro. The unfolding curves shown in Fig. 7 should be biphasic. However, because the protein was excited with 295 nm UV light, one of the domain unfoldings is invisible and the unfolding curve only registered the structural changes of the tryptophan-containing domain. W31 and W207/W218 thus report conformational changes of the N-terminal and C-terminal domains, respectively. It is then clear that the N-terminal domain is labile than the C-terminal domain. The stabilization of the catalytic N-terminal domain by the C-terminal domain III is thus confirmed. The intrinsic conformational stability of domain (I+II) without domain III has a much higher free energy (7.27 kcal mol–1) than full-length 3CLpro (1.22 kcal mol–1) (Table 2). The significant larger mGdnCl value of 3CLI+II (4.94 kcal mol–1 M–1) than full-length 3CLpro (1.15 kcal mol–1 M–1) clearly indicates the pronounced increase of surface area exposure on denaturation of 3CLI+II (26). This actually defines the functional role of domain III in SARS-CoV main protease.
After dissociation, the distribution of three structural forms of 3CLpro in various GdnCl concentrations is very clear. It includes a folded monomer, an intermediate with folded domain III but unfolded domain (I+II), and a completely unfolded form. The enzyme starts to dissociate at very early stage, at GdnCl concentration of <0.1 M. The dissociated monomer then starts to unfold at <0.4 M and reaches the first plateau at 2–3 M. The second unfolding then starts, which reaches another plateau at 4.5 M GdnCl. The complete dissociation and differential unfolding of the structural domains are summarized in Fig. 8. This schematic presentation only lists the major enzyme species of the complex folding process. The sharp unfolding curves for 3CLI+II (Fig. 6) and the completely reversible of the unfolding process (Fig. 2 B) consistent with that 3CLI+II without domain III can fold independently into an intact chymotrypsin-like fold. Domain III thus must have extra functional role.
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) have observed a nanochannel between domain III and the active center and proposed a regulatory effect of the extra domain III in relaying the catalytic machinery. Chen et al. (21) suggest that the enzyme follows an association-activation-catalysis-dissociation catalytic cycle. This will provide further insight into the functional role of domain III in the dimerization and involvement in the enzyme activity regulation beyond simple stabilization of the catalytic N-terminal domain. In summary, both 3CLI+II and 3CLpro show a completely reversible unfolding/refolding process in GdnCl. We demonstrate here a clear-cut case of the differential stability of quaternary and domain structures. The unfolding studies of SARS-CoV main protease may shed light on the inherent structural stability of this important viral enzyme.
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ACKNOWLEDGEMENTS |
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This work was supported by National Science Council, Republic of China (NSC 94-2320-B-010-060).
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FOOTNOTES |
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Submitted on June 19, 2006; accepted for publication November 2, 2006.
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