title: Nucleotide channel of RNA-dependent RNA polymerase used for intermolecular uridylylation of protein primer.
abstract: Poliovirus VPg is a 22 amino acid residue peptide that serves as the protein primer for replication of the viral RNA genome. VPg is known to bind directly to the viral RNA-dependent RNA polymerase, 3D, for covalent uridylylation, yielding mono and di-uridylylated products, VPg-pU and VPg-pUpU, which are subsequently elongated. To model the docking of the VPg substrate to a putative VPg-binding site on the 3D polymerase molecule, we performed a variety of structure-based computations followed by experimental verification. First, potential VPg folded structures were identified, yielding a suite of predicted beta-hairpin structures. These putative VPg structures were then docked to the region of the polymerase implicated by genetic experiments to bind VPg, using grid-based and fragment-based methods. Residues in VPg predicted to affect binding were identified through molecular dynamics simulations, and their effects on the 3D-VPg interaction were tested computationally and biochemically. Experiments with mutant VPg and mutant polymerase molecules confirmed the predicted binding site for VPg on the back side of the polymerase molecule during the uridylylation reaction, opposite to that predicted to bind elongating RNA primers.
authors.affiliation: Department of Biomedical Informatics, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Andres B
authors.initials: AB
authors.lastname: Tellez
authors.forename: Scott
authors.initials: S
authors.lastname: Crowder
authors.forename: Jeannie F
authors.initials: JF
authors.lastname: Spagnolo
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Thompson
authors.forename: Olve B
authors.initials: OB
authors.lastname: Peersen
authors.forename: Douglas L
authors.initials: DL
authors.lastname: Brutlag
authors.forename: Karla
authors.initials: K
authors.lastname: Kirkegaard
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D016679:Genome, Viral; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D017955:Poliovirus / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014529:Uridine / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D009711:Nucleotides; D012367:RNA, Viral; D012261:Ribonucleoproteins; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase; D014529:Uridine