title: Triphosphate Reorientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA Polymerases.
abstract: The nucleotide incorporation fidelity of the viral RNA-dependent RNA polymerase (RdRp) is important for maintaining functional genetic information but, at the same time, is also important for generating sufficient genetic diversity to escape the bottlenecks of the host's antiviral response. We have previously shown that the structural dynamics of the motif D loop are closely related to nucleotide discrimination. Previous studies have also suggested that there is a reorientation of the triphosphate of the incoming nucleotide, which is essential before nucleophilic attack from the primer RNA 3'-hydroxyl. Here, we have used31P NMR with poliovirus RdRp to show that the binding environment of the triphosphate is different when correctversusincorrect nucleotide binds. We also show that amino acid substitutions at residues known to interact with the triphosphate can alter the binding orientation/environment of the nucleotide, sometimes lead to protein conformational changes, and lead to substantial changes in RdRp fidelity. The analyses of other fidelity variants also show that changes in the triphosphate binding environment are not always accompanied by changes in the structural dynamics of the motif D loop or other regions known to be important for RdRp fidelity, including motif B. Altogether, our studies suggest that the conformational changes in motifs B and D, and the nucleoside triphosphate reorientation represent separable, "tunable" fidelity checkpoints.
authors.affiliation: From the Departments of Chemistry and.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Yang
authors.affiliation: From the Departments of Chemistry and.
authors.forename: Xinran
authors.initials: X
authors.lastname: Liu
authors.affiliation: From the Departments of Chemistry and.
authors.forename: Derek M
authors.initials: DM
authors.lastname: Musser
authors.affiliation: Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Ibrahim M
authors.initials: IM
authors.lastname: Moustafa
authors.affiliation: Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Jamie J
authors.initials: JJ
authors.lastname: Arnold
authors.affiliation: Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Craig E
authors.initials: CE
authors.lastname: Cameron
authors.affiliation: From the Departments of Chemistry and ddb12@psu.edu.
authors.forename: David D
authors.initials: DD
authors.lastname: Boehr
mesh_terms: D020816:Amino Acid Motifs; D020134:Catalytic Domain; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics*; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics; D011122:Polyphosphates / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D012367:RNA, Viral / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D009711:Nucleotides; D011122:Polyphosphates; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; C005692:triphosphoric acid
keywords: RNA polymerase; RNA virus; enzyme kinetics; enzyme mechanism; nuclear magnetic resonance (NMR); plus-stranded RNA virus; poliovirus; polymerase fidelity; protein dynamic