PDBsum entry 3ee7

Viral protein

PDB id: 3ee7

Contents

Protein chains

106 a.a. *

115 a.a. *

Ligands

PO4 ×8

GOL ×16

Waters ×116

* Residue conservation analysis

PDB id:

3ee7

Name:

Viral protein

Title:

Crystal structure of sars-cov nsp9 g104e

Structure:

Replicase polyprotein 1a. Chain: a, b, c, d. Synonym: pp1a, orf1a polyprotein, non-structural protein 1, nsp1, leader protein, non-structural protein 2, nsp2, p65 homolog, non- structural protein 3, nsp3, papain-like proteinase, pl-pro, pl2-pro, non-structural protein 4, nsp4, 3c-like proteinase, 3cl-pro, 3clp, nsp5, non-structural protein 6, nsp6, non-structural protein 7, nsp7, non-structural protein 8, nsp8, non-structural protein 9, nsp9, non- structural protein 10, nsp10, growth factor-like peptide, gfl, non-

Source:

Sars coronavirus. Sars-cov. Organism_taxid: 227859. Strain: urbani. Gene: 1a. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.60Å R-factor: 0.216 R-free: 0.268

Authors:

Z.J.Miknis,E.F.Donaldson,T.C.Umland,R.Rimmer,R.S.Baric,L.W.Schultz

Key ref:

Z.J.Miknis et al. (2009). Severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth. J Virol, 83, 3007-3018. PubMed id: 19153232

Date:

04-Sep-08 Release date: 24-Mar-09

Protein chain

P0C6U8  (R1A_CVHSA) -  Replicase polyprotein 1a from Severe acute respiratory syndrome coronavirus

Seq: 4382 a.a.

Struc: 106 a.a.*

Protein chains

P0C6U8  (R1A_CVHSA) -  Replicase polyprotein 1a from Severe acute respiratory syndrome coronavirus

Seq: 4382 a.a.

Struc: 115 a.a.*

* PDB and UniProt seqs differ at 8 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: Chains A, B, C, D: 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 2: Chains A, B, C, D: 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 3: Chains A, B, C, D: E.C.3.4.22.- - ?????
• Enzyme class 4: Chains A, B, C, D: E.C.3.4.22.69 - Sars coronavirus main proteinase.

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

Added reference

J Virol 83:3007-3018 (2009)

PubMed id: 19153232

Severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth.

Z.J.Miknis, E.F.Donaldson, T.C.Umland, R.A.Rimmer, R.S.Baric, L.W.Schultz.

ABSTRACT

The severe acute respiratory syndrome coronavirus (SARS-CoV) devotes a significant portion of its genome to producing nonstructural proteins required for viral replication. SARS-CoV nonstructural protein 9 (nsp9) was identified as an essential protein with RNA/DNA-binding activity, and yet its biological function within the replication complex remains unknown. Nsp9 forms a dimer through the interaction of parallel alpha-helices containing the protein-protein interaction motif GXXXG. In order to study the role of the nsp9 dimer in viral reproduction, residues G100 and G104 at the helix interface were targeted for mutation. Multi-angle light scattering measurements indicated that G100E, G104E, and G104V mutants are monomeric in solution, thereby disrupting the dimer. However, electrophoretic mobility assays revealed that the mutants bound RNA with similar affinity. Further experiments using fluorescence anisotropy showed a 10-fold reduction in RNA binding in the G100E and G104E mutants, whereas the G104V mutant had only a 4-fold reduction. The structure of G104E nsp9 was determined to 2.6-A resolution, revealing significant changes at the dimer interface. The nsp9 mutations were introduced into SARS-CoV using a reverse genetics approach, and the G100E and G104E mutations were found to be lethal to the virus. The G104V mutant produced highly debilitated virus and eventually reverted back to the wild-type protein sequence through a codon transversion. Together, these data indicate that dimerization of SARS-CoV nsp9 at the GXXXG motif is not critical for RNA binding but is necessary for viral replication.