PDBsum entry 4hdh

Transferase

PDB id: 4hdh

Contents

Protein chains

613 a.a.

Ligands

ATP ×2

Metals

_ZN ×4

Waters ×419

PDB id:

4hdh

Name:

Transferase

Title:

Crystal structure of viral rdrp in complex with atp

Structure:

Polyprotein. Chain: a, b. Fragment: RNA dependent RNA polymerase module, residues 272-905. Engineered: yes

Source:

Japanese encephalitis virus. Organism_taxid: 11072. Strain: p20778. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.28Å R-factor: 0.228 R-free: 0.272

Authors:

P.Surana,D.T.Nair

Key ref:

P.Surana et al. (2014). RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state. Nucleic Acids Res, 42, 2758-2773. PubMed id: 24293643 DOI: 10.1093/nar/gkt1106

Date:

02-Oct-12 Release date: 25-Dec-13

Protein chains

P27395  (POLG_JAEV1) -  Genome polyprotein from Japanese encephalitis virus (strain SA-14)

Seq: 3432 a.a.

Struc: 613 a.a.*

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: 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

5'-end (5'-triphosphoguanosine)-ribonucleoside in mRNA + S-adenosyl-L- methionine = 5'-end (N(7)-methyl 5'-triphosphoguanosine)-ribonucleoside in mRNA + S-adenosyl-L-homocysteine (Bound ligand (Het Group name = ATP) matches with 46.15% similarity)

• Enzyme class 2: 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⁺

5'-end (N(7)-methyl 5'-triphosphoguanosine)-ribonucleoside in mRNA + S-adenosyl-L-methionine = 5'-end (N(7)-methyl 5'-triphosphoguanosine)- (2'-O-methyl-ribonucleoside) in mRNA + S-adenosyl-L-homocysteine + H(+) (Bound ligand (Het Group name = ATP) matches with 46.15% similarity)

• Enzyme class 3: E.C.2.7.7.48 - RNA-directed Rna polymerase.
• Reaction: RNA(n) + a ribonucleoside 5'-triphosphate = RNA(n+1) + diphosphate
• Enzyme class 4: E.C.3.4.21.91 - flavivirin.
• Reaction: Selective hydrolysis of Xaa-Xaa-|-Xbb bonds in which each of the Xaa can be either Arg or Lys and Xbb can be either Ser or Ala.
• Enzyme class 5: E.C.3.6.1.15 - nucleoside-triphosphate phosphatase.
• Reaction: a ribonucleoside 5'-triphosphate + H2O = a ribonucleoside 5'-diphosphate + phosphate + H⁺
• Enzyme class 6: E.C.3.6.4.13 - Rna helicase.
• Reaction: ATP + H2O = ADP + phosphate + H⁺

ATP (Bound ligand (Het Group name = ATP) corresponds exactly) + H2O = ADP + phosphate + H(+)

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: 10.1093/nar/gkt1106

Nucleic Acids Res 42:2758-2773 (2014)

PubMed id: 24293643

RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state.

P.Surana, V.Satchidanandam, D.T.Nair.

ABSTRACT

Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5'-CU-3' at the 3'-end of the flaviviral genome is highly conserved. Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRpGTP structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRpGTP structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn(2+) ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a checkpoint to prevent erroneous template-independent RNA synthesis by jRdRp during initiation.