3C-like Proteinase from SARS Coronavirus Catalyzes Substrate Hydrolysis by a General Base Mechanism

Severe acute respiratory syndrome (SARS) coronavirus is a novel human coronavirus and is responsible for SARS infection. SARS coronavirus 3C-like proteinase (SARS 3CLpro ) plays key roles in viral replication and transcription and is an attractive target for anti-SARS drug discovery. In this report, we quantitatively characterized the dimerization features of the full-length and N-terminal residues 1-7 deleted SARS 3CL pro s by using glutaraldehyde cross-linking SDS-PAGE, size-exclusion chromatography, and isothermal titration calorimeter techniques. Glutaraldehyde cross-linking SDS-PAGE and size-exclusion chromatography results show that, similar to the full-length SARS 3CLpro , the N-terminal deleted SARS 3CL pro still remains a dimer/monomer mixture within a wide range of protein concentrations. Isothermal titration calorimeter determinations indicate that the equilibrium dissociation constant (K d ) of the N-terminal deleted proteinase dimer (262 M) is very similar to that of the full-length proteinase dimer (227 M). Enzymatic activity assay using the fluorescence resonance energy transfer method reveals that N-terminal deletion results in almost complete loss of enzymatic activity for SARS 3CL pro . Molecular dynamics and docking simulations demonstrate the N-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer, which increases the angle between the two protomers and reduces the binding pocket that is not beneficial to the substrate binding. This conclusion is verified by the surface plasmon resonance biosensor determination, indicating that the model substrate cannot bind to the N-terminal deleted proteinase. These results suggest the N terminus is not indispensable for the proteinase dimerization but may fix the dimer at the active state and is therefore vital to enzymatic activity.

show abstract

“…S2). This is in agreement with the published data (13,26), further indicating that our fluorogenic method for determining SARS 3CL…”

Section: Folding Behavior and Thermal Stability Of The N-terminalsupporting

confidence: 92%

Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase N Terminus Is Indispensable for Proteolytic Activity but Not for Enzyme Dimerization

Chen¹,

Chen²,

Tan³

et al. 2005

Journal of Biological Chemistry

123

View full text Add to dashboard Cite

show abstract

“…Enzymatic Activity of the Wild-type and the Hybrid SARS 3CL pro -In the previous study, we have reported that the active site mutation C145A gave an inactive enzyme (28). The mutant did not show any visible enzyme activity at concentrations as high as 100 M. However, when it was added into the low concentration solution of the wild-type enzyme (protein was partly in monomeric form), the resulting hybrid enzyme exhibited increased activity (Fig.…”

Section: Resultsmentioning

confidence: 85%

“…Colorimetric Enzyme Assay for the Wild-type and the Hybrid Protein-The enzyme activity was measured by a colorimetric assay as reported previously (28). In short, 20 l of pNA substrate stock solution (2 mM Thr-Ser-Ala-Val-Leu-Gln-pNA water solution) was added to 180 l of 25°C preheated reaction buffer (40 mM phosphate-buffered saline, 1 mM EDTA, 0.2 mg/ml bovine serum albumin, 3 mM dithiothreitol, pH 7.3), which contained SARS 3CL pro wild-type enzyme and the C145A mutant.…”

Section: Site-directed Mutagenesis Of Sars 3clmentioning

confidence: 99%

Only One Protomer Is Active in the Dimer of SARS 3C-like Proteinase

Chen

Wei

Huang

et al. 2006

Journal of Biological Chemistry

Self Cite

129

155

View full text Add to dashboard Cite

The severe acute respiratory syndrome coronavirus 3C-like protease has been proposed to be a key target for structurally based drug design against SARS. The enzyme exists as a mixture of dimer and monomer, and only the dimer was considered to be active. In this report, we have investigated, using molecular dynamics simulation and mutational studies, the problems as to why only the dimer is active and whether both of the two protomers in the dimer are active. The molecular dynamics simulations show that the monomers are always inactive, that the two protomers in the dimer are asymmetric, and that only one protomer is active at a time. The enzyme activity of the hybrid severe acute respiratory syndrome coronavirus 3C-like protease of the wild-type protein and the inactive mutant proves that the dimerization is important for enzyme activity and only one active protomer in the dimer is enough for the catalysis. Our simulations also show that the right conformation for catalysis in one protomer can be induced upon dimer formation. These results suggest that the enzyme may follow the association, activation, catalysis, and dissociation mechanism for activity control.In early 2003, a highly epidemic disease named severe acute respiratory syndrome (SARS) 3 first broke out in China and then quickly spread to other circumjacent countries (1). Research proved that the nosogenesis was a novel coronavirus. In the coronavirus life cycle, 3C-like proteinase (3CL pro ) is important and indispensable and is a pivotal target in anti-SARS drug design (2). SARS 3CL pro shares 40 and 44% sequence identity to 3CL pro of human coronaviruses 229E and transmissible gastroenteritis virus, the crystal structures of which have been resolved (2, 3). Several homology models for SARS 3CL pro have been reported (2, 4, 5). More recently, the crystal structures of the enzyme and the inhibitor-enzyme complex have been determined (6 -11). All structures are very similar and consist of three domains. The first two domains form a chymotrypsin fold, and the third domain is an extra helix domain that plays an important role in dimerization and enzyme activity (12). All of the proteins are dimeric in the crystal structures, and there exists an equilibrium between the monomer and dimer in solution. In our previous work, we have observed that the activity increases with the increase of enzyme concentration, indicating the dimer is the active form of the proteinase (13). Other groups have studied the function of the N-finger in dimerization and enzyme activity. The N-terminal residues 1-5 delete transmissible gastroenteritis virus 3CL pro , and the N-terminal residues 1-7 delete SARS 3CL pro ; both have been reported to have no enzyme activities (3, 14 -16). Interestingly, Chen et al. (14) report that the N-finger deletion mutation does not affect the dimerization of SARS 3CLpro . Contrary to this, Hsu et al. (16) has found that the N-4 truncated protease is mainly monomeric and has little enzyme activity, but the N-3 truncated protease is almost the ...

show abstract

“…Therefore, the search for more effective and potent antivirals for the SARS virus is of current interest. Recent identification of the viral genome (17)(18)(19), the viral receptor (20), the viral main protease (the chymotrypsin-like protease, also called 3CL protease) and its structure (21,22), and activity studies (23,24) have provided a better understanding of this devastating disease and should facilitate the development of effective therapeutic agents. This report describes a cell-based assay using SARS-CoV and Vero E6 cells (18) to screen a collection of nearly 10,000 compounds and natural products to identify antiviral agents for SARS.…”

mentioning

confidence: 99%

Small molecules targeting severe acute respiratory syndrome human coronavirus

Jan

Hwa

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

493

416

View full text Add to dashboard Cite

Severe acute respiratory syndrome (SARS) is an infectious disease caused by a novel human coronavirus. Currently, no effective antiviral agents exist against this type of virus. A cell-based assay, with SARS virus and Vero E6 cells, was developed to screen existing drugs, natural products, and synthetic compounds to identify effective anti-SARS agents. Of >10,000 agents tested, Ϸ50 compounds were found active at 10 M; among these compounds, two are existing drugs (Reserpine 13 and Aescin 5) and several are in clinical development. These 50 active compounds were tested again, and compounds 2-6, 10, and 13 showed active at 3 M. The 50% inhibitory concentrations for the inhibition of viral replication (EC50) and host growth (CC50) were then measured and the selectivity index (SI ‫؍‬ CC50͞EC50) was determined. The EC50, based on ELISA, and SI for Reserpine, Aescim, and Valinomycin are 3.4 M (SI ‫؍‬ 7.3), 6.0 M (SI ‫؍‬ 2.5), and 0.85 M (SI ‫؍‬ 80), respectively. Additional studies were carried out to further understand the mode of action of some active compounds, including ELISA, Western blot analysis, immunofluorescence and flow cytometry assays, and inhibition against the 3CL protease and viral entry. Of particular interest are the two anti-HIV agents, one as an entry blocker and the other as a 3CL protease inhibitor (Ki ‫؍‬ 0.6 M).

show abstract

3C-like Proteinase from SARS Coronavirus Catalyzes Substrate Hydrolysis by a General Base Mechanism

Cited by 209 publications

References 32 publications

Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase N Terminus Is Indispensable for Proteolytic Activity but Not for Enzyme Dimerization

Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase N Terminus Is Indispensable for Proteolytic Activity but Not for Enzyme Dimerization

Only One Protomer Is Active in the Dimer of SARS 3C-like Proteinase

Small molecules targeting severe acute respiratory syndrome human coronavirus

Contact Info

Product

Resources

About