4R0E

Crystal Structure of the Poliovirus RNA-Dependent RNA Polymerase Low-Fidelity Mutant 3Dpol H273R

Summary for 4R0E

• Entry DOI: 10.2210/pdb4r0e/pdb
• Related: 2IJF
• Descriptor: RNA-directed RNA polymerase (2 entities in total)
• Functional Keywords: rna-dependent rna polymerase, fidelity, low-fidelity, rna polymerase, poliovirus, transferase
• Biological source: Human poliovirus 1
• Cellular location: Capsid protein VP0: Virion. Capsid protein VP4: Virion. Capsid protein VP2: Virion. Capsid protein VP3: Virion. Capsid protein VP1: Virion. Protein 2B: Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side . Protein 2C: Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side . Protein 3A: Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side . Protein 3AB: Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side . Viral protein genome-linked: Virion. Protease 3C: Host cytoplasm. Protein 3CD: Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side . RNA-directed RNA polymerase: Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side : P03300
• Total number of polymer chains: 1
• Total formula weight: 52590.98

Authors

Marcotte, L.L.,Gohara, D.W.,Filman, D.J.,Hogle, J.M. (deposition date: 2014-07-30, release date: 2014-11-12, Last modification date: 2023-09-20)

Primary citation

Moustafa, I.M.,Korboukh, V.K.,Arnold, J.J.,Smidansky, E.D.,Marcotte, L.L.,Gohara, D.W.,Yang, X.,Sanchez-Farran, M.A.,Filman, D.,Maranas, J.K.,Boehr, D.D.,Hogle, J.M.,Colina, C.M.,Cameron, C.E.
Structural Dynamics as a Contributor to Error-prone Replication by an RNA-dependent RNA Polymerase.
J.Biol.Chem., 289:36229-36248, 2014

PubMed Abstract: RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity.

PubMed: 25378410
DOI: 10.1074/jbc.M114.616193

Experimental method

X-RAY DIFFRACTION (3 Å)