PDBsum entry 2rhb

Viral protein

PDB id: 2rhb

Contents

Protein chains

(+ 0 more) 345 a.a. *

Waters ×441

* Residue conservation analysis

PDB id:

2rhb

Name:

Viral protein

Title:

Crystal structure of nsp15-h234a mutant- hexamer in asymmetric unit

Structure:

Uridylate-specific endoribonuclease. Chain: a, b, c, d, e, f. Engineered: yes. Mutation: yes

Source:

Sars coronavirus. Organism_taxid: 227859. Strain: urbani isolate of sars-cov. Gene: nsp15. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_cell_line: bl21 star.

Resolution:

2.80Å R-factor: 0.200 R-free: 0.260

Authors:

S.Palaninathan,K.Bhardwaj,J.M.O.Alcantara,L.Guarino,L.L.Yi,C.C.Kao, J.Sacchettini

Key ref:

K.Bhardwaj et al. (2008). Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease nsp15. J Biol Chem, 283, 3655-3664. PubMed id: 18045871 DOI: 10.1074/jbc.M708375200

Date:

08-Oct-07 Release date: 27-Nov-07

Protein chains

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

Seq: 7073 a.a.

Struc: 345 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

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: 10.1074/jbc.M708375200

J Biol Chem 283:3655-3664 (2008)

PubMed id: 18045871

Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease nsp15.

K.Bhardwaj, S.Palaninathan, J.M.Alcantara, L.Li Yi, L.Guarino, J.C.Sacchettini, C.C.Kao.

ABSTRACT

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3' of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.

Selected figure(s)

Figure 1.
FIGURE 1. Structure of SARS-CoV Nsp15. A, surface representation of a subunit showing three domains. Active site residues are colored by element and indicated by an arrow. B, arrangement of six subunits from top view of hexamer. N-terminal, middle, and C-terminal domains are indicated as N, M, and C, respectively. The arrows indicate the positions of catalytic sites in top trimer. C, side view of hexamer showing arrangement and N- to N-terminal interaction of the top trimer (T1) with bottom trimer (T2). Six subunits are labeled as a-c and are colored as follows: T1a, pink; T1b, blue; T1c, cyan; T2a, red; T2b, green; T2c, golden. This color scheme is used throughout the figures. Catalytic residues are colored yellow. D, worm diagram drawn based on b-factor. Worm thickness is directly proportional to flexibility, i.e. the thickest region indicates most flexible. E, overlap of the catalytic residues within the active sites of the six subunits. The structures are anchored by the backbond of His^249.

Figure 2.
FIGURE 2. Subunit interaction. A, wire diagram of subunit T1a and its interaction with other subunits. Contact regions are boxed and labeled (boxes B-E). The inset shows molecular arrangement and atomic distances between E3 and L2 (left) and the gel filtration profile of the E3A mutant of Nsp15 (20). H, T, and M denote positions corresponding to elution volume of a hexamer, trimer, and monomer of Nsp15, respectively. B-E, contacting residues and the calculated distances between them (left). Atomic distances are calculated using Chimera. Gel filtration chromatography elution profiles of the indicated mutant proteins (right). The values below the mutant name refer to the cleavage rate relative to WT in parentheses.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 3655-3664) copyright 2008.

Figures were selected by an automated process.