title: Identification of two nucleotide-binding domains on the PB1 subunit of influenza virus RNA polymerase.
abstract: Influenza virus RNA polymerase is a multifunctional and multisubunit enzyme consisting of three viral P proteins, PB1, PB2, and PA. We have previously shown that radioactive 8-azido GTP (8-N3 GTP) was photo-crosslinked specifically to the PB1 subunit. Here we confirmed the specific crosslinking of PB1 using oxidized GTP and further identified the GTP analogue binding domains after proteolytic cleavage of the crosslinked PB1 with V8 protease. The cleavage pattern of PB1 was determined by analysis of the amino-terminal proximal sequence of fragments generated in the presence of increasing concentrations of V8 protease. The GTP-crosslinking was identified in three fragments: two adjacent fragments, P6 starting from residue 179 and Pllb starting from residue 298; and the third fragment, P11c, starting from residue 458. Thus, we propose that two GTP-binding sites exist in the PB1 subunit, i.e., the amino terminal-proximal site I located at the boundary between P6 and Pllb, and the carboxy terminal proximal site II on P11c fragment. The locations of GTP-binding sites I and II are close to those of sequence motif A and motif D, respectively, conserved among RNA-dependent RNA polymerases. Of the two fragments forming site I, the crosslinking of 8-N3 GTP is higher to P11b, while that of oxidized GTP is higher to P6, suggesting that the ribose and guanine moieties of GTP bound in this binding pocket face P6 and P11c, respectively. From the V8 concentration dependent change in proteolytic cleavage pattern, it is likely that the two GTP-binding sites on PB1 protein are located on structurally different domains. The existence of two GTP-binding sites is discussed in relation to the binding sites for substrates, primers, and products.
authors.affiliation: Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka.
authors.forename: Y
authors.initials: Y
authors.lastname: Asano
authors.forename: A
authors.initials: A
authors.lastname: Ishihama
mesh_terms: D000595:Amino Acid Sequence; D001386:Azides / Q000032:analysis / Q000378:metabolism; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000032:analysis / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*; D009980:Influenza A virus / Q000201:enzymology*; D046911:Macromolecular Substances; D010447:Peptide Hydrolases / Q000378:metabolism; D010449:Peptide Mapping; D012697:Serine Endopeptidases / Q000378:metabolism / Q000494:pharmacology; D014764:Viral Proteins / Q000032:analysis / Q000378:metabolism*
chemical_list: C021961:8-azidoguanosine triphosphate; D001386:Azides; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D014764:Viral Proteins; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase