Structural Dynamics as a Contributor to Error-prone Replication by a RNA-dependent RNA Polymerase [Enzymology]

November 6th, 2014 by 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.

RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRps) 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 have used X-ray crystallography, molecular dynamics simulations (MD), NMR spectroscopy and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type enzyme (WT). We show that the nucleotide-binding site toggles between nucleotide-binding-occluded and nucleotide-binding-competent states. The conformational dynamics between these states were enhanced by binding to primed-template RNA. For 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 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.