PDBsum entry 2hob

Viral protein

PDB id: 2hob

Contents

Protein chain

306 a.a. *

Ligands

02J-ALA-VAL-LEU-PJE-010

Waters ×308

* Residue conservation analysis

PDB id:

2hob

Name:

Viral protein

Title:

Crystal structure of sars-cov main protease with authentic n and c- termini in complex with a michael acceptor n3

Structure:

Replicase polyprotein 1ab. Chain: a. Fragment: 3c-like proteinase. Engineered: yes. N-[(5-methylisoxazol-3-yl)carbonyl]alanyl-l-valyl-n~1~- ((1r,2z)-4-(benzyloxy)-4-oxo-1-{[(3r)-2-oxopyrrolidin-3- yl]methyl}but-2-enyl)-l-leucinamide. Chain: b. Engineered: yes

Source:

Sars coronavirus. Organism_taxid: 227859. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Synthetic construct. Organism_taxid: 32630

Resolution:

1.95Å R-factor: 0.202 R-free: 0.221

Authors:

X.Xue,H.Yang,W.Shen,Q.Zhao,J.Li,Z.Rao

Key ref:

X.Xue et al. (2007). Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction. J Mol Biol, 366, 965-975. PubMed id: 17189639 DOI: 10.1016/j.jmb.2006.11.073

Date:

14-Jul-06 Release date: 03-Apr-07

Protein chain

P0C6X7  (R1AB_CVHSA) -  Replicase polyprotein 1ab from Severe acute respiratory syndrome coronavirus

Seq: 7073 a.a.

Struc: 306 a.a.

Enzyme reactions

• Enzyme class 2: E.C.2.1.1.- - ?????
• Enzyme class 3: E.C.2.1.1.56 - mRNA (guanine-N(7))-methyltransferase.
• Reaction: a 5'-end (5'-triphosphoguanosine)-ribonucleoside in mRNA + S-adenosyl-L- methionine = a 5'-end (N(7)-methyl 5'-triphosphoguanosine)-ribonucleoside in mRNA + S-adenosyl-L-homocysteine
• Enzyme class 4: E.C.2.1.1.57 - methyltransferase cap1.
• Reaction: a 5'-end (N(7)-methyl 5'-triphosphoguanosine)-ribonucleoside in mRNA + S-adenosyl-L-methionine = a 5'-end (N(7)-methyl 5'-triphosphoguanosine)- (2'-O-methyl-ribonucleoside) in mRNA + S-adenosyl-L-homocysteine + H⁺
• Enzyme class 5: E.C.2.7.7.48 - RNA-directed Rna polymerase.
• Reaction: RNA(n) + a ribonucleoside 5'-triphosphate = RNA(n+1) + diphosphate
• Enzyme class 6: E.C.2.7.7.50 - mRNA guanylyltransferase.
• Reaction: a 5'-end diphospho-ribonucleoside in mRNA + GTP + H⁺ = a 5'-end (5'-triphosphoguanosine)-ribonucleoside in mRNA + diphosphate
• Enzyme class 7: E.C.3.1.13.- - ?????
• Enzyme class 8: E.C.3.4.19.12 - ubiquitinyl hydrolase 1.
• Reaction: Thiol-dependent hydrolysis of ester, thiolester, amide, peptide and isopeptide bonds formed by the C-terminal Gly of ubiquitin (a 76-residue protein attached to proteins as an intracellular targeting signal).
• Enzyme class 9: E.C.3.4.22.- - ?????
• Enzyme class 10: E.C.3.4.22.69 - Sars coronavirus main proteinase.
• Enzyme class 11: E.C.3.6.4.12 - Dna helicase.
• Reaction: ATP + H2O = ADP + phosphate + H⁺
• Enzyme class 12: E.C.3.6.4.13 - Rna helicase.
• Reaction: ATP + H2O = ADP + phosphate + H⁺
• Enzyme class 13: E.C.4.6.1.- - ?????

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2006.11.073

J Mol Biol 366:965-975 (2007)

PubMed id: 17189639

Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction.

X.Xue, H.Yang, W.Shen, Q.Zhao, J.Li, K.Yang, C.Chen, Y.Jin, M.Bartlam, Z.Rao.

ABSTRACT

The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (M(pro)) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV M(pro) was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV M(pro) with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV M(pro) with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of M(pro) are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV M(pro) and its complex with a Michael acceptor inhibitor were determined to 1.6 Angstroms and 1.95 Angstroms resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV M(pro) could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV M(pro) is more appropriate for mechanistic characterization and inhibitor design.

Selected figure(s)

Figure 3.
Figure 3. Superposition of the S1 pockets of GPLGS-WT and WT SARS-CoV M^pro (in stereo). (a) Superposition of the S1 pockets in protomer A of GPLGS-WT and that of protomer A* of WT SARS-CoV M^pro. Protomer A* of WT is in blue; protomer A of GPLGS-WT is in yellow; protomer B* of WT is in magenta; protomer B of GPLGS-WT is in red. In the WT structure, the amino group (NH[2]) of Ser1 in protomer B* donates a 3.0 Å hydrogen bond to the carboxylate group of Glu166 and a 2.7 Å hydrogen bond to the main-chain carbonyl group of Phe140 in protomer A*, stabilizing the S1 pocket. The NH of Gly143 moves 0.8 Å toward the activity site; the main chain of residues 142-143 moves toward the S1 subsite; the side-chain of Asn-A*142 flips over with a 6 Å shift compared with protomer A of GPLGS-WT. (b) Superposition of the S1 pockets in protomer B of GPLGS-WT and that of Protomer A* of WT SARS-CoV M^pro. Protomer A* of WT is in blue; protomer B of GPLGS-WT is in yellow; protomer B* of WT is in magenta; protomer A of GPLGS-WT is in red. The S1 pocket of protomer B collapses partly with reorientation of Glu166 and residues 140–143. No electron density was visible for residues A1 and A2. Figure 3. Superposition of the S1 pockets of GPLGS-WT and WT SARS-CoV M^pro (in stereo). (a) Superposition of the S1 pockets in protomer A of GPLGS-WT and that of protomer A* of WT SARS-CoV M^pro. Protomer A* of WT is in blue; protomer A of GPLGS-WT is in yellow; protomer B* of WT is in magenta; protomer B of GPLGS-WT is in red. In the WT structure, the amino group (NH[2]) of Ser1 in protomer B* donates a 3.0 Å hydrogen bond to the carboxylate group of Glu166 and a 2.7 Å hydrogen bond to the main-chain carbonyl group of Phe140 in protomer A*, stabilizing the S1 pocket. The NH of Gly143 moves 0.8 Å toward the activity site; the main chain of residues 142-143 moves toward the S1 subsite; the side-chain of Asn-A*142 flips over with a 6 Å shift compared with protomer A of GPLGS-WT. (b) Superposition of the S1 pockets in protomer B of GPLGS-WT and that of Protomer A* of WT SARS-CoV M^pro. Protomer A* of WT is in blue; protomer B of GPLGS-WT is in yellow; protomer B* of WT is in magenta; protomer A of GPLGS-WT is in red. The S1 pocket of protomer B collapses partly with reorientation of Glu166 and residues 140–143. No electron density was visible for residues A1 and A2.

Figure 4.
Figure 4. Differences between the complex structures of WT and GPLGS-WT. (a) Inhibitor N3. (b) Superposition of the substrate-binding pockets in protomer A of GPLGS-WT and that in protomer A* of WT. In the WT-N3 complex structure, the NH[2] group of Ser1 in protomer B* was still hydrogen-bonded to the carboxylate group of Glu166 and the carbonyl group of Phe140 in protomer A*, stabilizing the S1 pocket. In the GPLGS-WT-N3 complex structure, however, the two hydrogen bonds described above were not found. Instead, an ordered water molecule was observed in the S1 pocket. Protomer A* of WT is in blue; protomer A of GPLGS-WT is in yellow; inhibitor N3 (complexed with WT) is in magenta; inhibitor N3 (complexed with GPLGS-WT) is in red; protomer B* of WT is in green; protomer B of GPLGS-WT is in cyan. Figure 4. Differences between the complex structures of WT and GPLGS-WT. (a) Inhibitor N3. (b) Superposition of the substrate-binding pockets in protomer A of GPLGS-WT and that in protomer A* of WT. In the WT-N3 complex structure, the NH[2] group of Ser1 in protomer B* was still hydrogen-bonded to the carboxylate group of Glu166 and the carbonyl group of Phe140 in protomer A*, stabilizing the S1 pocket. In the GPLGS-WT-N3 complex structure, however, the two hydrogen bonds described above were not found. Instead, an ordered water molecule was observed in the S1 pocket. Protomer A* of WT is in blue; protomer A of GPLGS-WT is in yellow; inhibitor N3 (complexed with WT) is in magenta; inhibitor N3 (complexed with GPLGS-WT) is in red; protomer B* of WT is in green; protomer B of GPLGS-WT is in cyan.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 366, 965-975) copyright 2007.

Figures were selected by an automated process.