Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance.
The fidelity of the poliovirus RNA-dependent RNA polymerase (3D(pol)) plays a direct role in the genomic evolution and pathogenesis of the virus. A single site mutation (Gly64Ser) that is remote from the catalytic center results in a higher fidelity polymerase. NMR studies with [methyl-(13)C]methionine-labeled protein were used to compare the solution structure and dynamics of wild-type and Gly64Ser 3D(pol). The chemical shifts for the Met6 resonance were significantly different between wild-type and Gly64Ser 3D(pol) when bound in ternary complexes with RNA and incorrect, but not with correct, nucleotide, suggesting that the Gly64Ser mutation induces structural changes in the N-terminal β-strand when the enzyme is bound to incorrect but not correct nucleotide. We also observe changes in the transverse relaxation times for methionines near regions important for nucleotide and RNA binding and catalysis. Our strategy to assign the [methyl-(13)C]methionine resonances involved separately mutating each of the 17 methionines. Several substitutions produced additional resonances for both Met6 and Met187, a reporter for RNA binding, and conformational changes in the highly conserved motif B loop, even though these methionines are greater than 20 Å apart. The results for Gly64Ser and the other mutants are intriguing considering that they can result in structural and/or dynamic changes to methionines distant from the site of mutation. We propose that there is a long-distance network operating throughout 3D(pol) that coordinates ligand binding, conformational changes, and catalysis. Mutation of Gly64 results in structural and/or dynamic changes to the network that may affect polymerase fidelity.
Yang
Xiaorong
X
Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
Welch
Jesse L
JL
Arnold
Jamie J
JJ
Boehr
David D
DD
D002384:Catalysis; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry; D020125:Mutation, Missense; D009711:Nucleotides / Q000378:metabolism; D017955:Poliovirus / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D008024:Ligands; D009711:Nucleotides; D008715:Methionine; D012324:RNA-Dependent RNA Polymerase