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Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance.

ABSTRACT: 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 ?-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.

SUBMITTER: Yang X

PROVIDER: S-EPMC2989882 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance.

Yang Xiaorong X Welch Jesse L JL Arnold Jamie J JJ Boehr David D DD

Biochemistry 20101101 43

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-typ ...[more]

PMID: 20860410

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Project description:BackgroundPutative high mutation rates of RNA viruses are believed to mediate undesirable phenomena, such as emergence of drug resistance. However, very little is known about biochemical fidelity rates for viral RNA-dependent RNA polymerases. Using a recently developed in vitro polymerase assay for poliovirus polymerase 3Dpol [Arnold and Cameron (2000) JBC 275:5329], we measured fidelity for each possible mismatch. Polymerase fidelity, in contrast to sequence error rate, is biochemically defined as kpol/Kd of {(correct plus incorrect) divided by incorrect} incorporations, such that a larger value connotes higher fidelity.ResultsTo derive kpol/Kd for correct base incorporation, we performed conventional pre-steady state single turnover measurements, yielding values that range from 0.62 to 9.4 microM-1 sec-1. Pre-steady state measurements for incorrect base incorporation were less straightforward: several anomalous phenomena interfered with data collection. To obtain pre-steady state kinetic data for incorrect base incorporation, three strategies were employed. (1) For some incorrect bases, a conventional approach was feasible, although care was taken to ensure that only single turnovers were being assessed. (2) Heparin or unlabeled RNA traps were used to simulate single turnover conditions. (3) Finally, for some incorrect bases, incorporation was so poor that single datapoints were used to provide kinetic estimates. Overall, we found that fidelity for poliovirus polymerase 3Dpol ranges from 1.2 x 10(4) to 1.0 x 10(6) for transition mutations and 3.2 x 10(5) to 4.3 x 10(7) for transversion mutations.ConclusionThese values are unexpectedly high showing that high RNA virus sequence variation is not due to intrinsically low polymerase fidelity. Based on unusual enzyme behavior that we observed, we speculate that RNA mismatches either directly or indirectly cause enzyme RNA dissociation. If so, high sequence variation of RNA viruses may be due to template-switch RNA recombination and/or unknown fitness/selection phenomena. These findings may lead to a mechanistic understanding of RNA virus error catastrophe and improved anti-viral strategies.

Project description:Bifidobacteria are widely used as probiotics in several commercial products; however, to date there is little knowledge about their carbohydrate metabolic pathways. In this work, we studied the metabolism of glucose and lactose in the widely used probiotic strain Bifidobacterium animalis subsp. lactis BB-12 by in vivo (13)C nuclear magnetic resonance (NMR) spectroscopy. The metabolism of [1-(13)C]glucose was characterized in cells grown in glucose as the sole carbon source. Moreover, the metabolism of lactose specifically labeled with (13)C on carbon 1 of the glucose or the galactose moiety was determined in suspensions of cells grown in lactose. These experiments allowed the quantification of some intermediate and end products of the metabolic pathways, as well as determination of the consumption rate of carbon sources. Additionally, the labeling patterns in metabolites derived from the metabolism of glucose specifically labeled with (13)C on carbon 1, 2, or 3 in cells grown in glucose or lactose specifically labeled in carbon 1 of the glucose moiety ([1-(13)Cglucose]lactose), lactose specifically labeled in carbon 1 of the galactose moiety ([1-(13)Cgalactose]lactose), and [1-(13)C]glucose in lactose-grown cells were determined in cell extracts by (13)C NMR. The NMR analysis showed that the recovery of carbon was fully compatible with the fructose 6-phosphate, or bifid, shunt. The activity of lactate dehydrogenase, acetate kinase, fructose 6-phosphate phosphoketolase, and pyruvate formate lyase differed significantly between glucose and lactose cultures. The transcriptional analysis of several putative glucose and lactose transporters showed a significant induction of Balat_0475 in the presence of lactose, suggesting a role for this protein as a lactose permease. This report provides the first in vivo experimental evidence of the metabolic flux distribution in the catabolic pathway of glucose and lactose in bifidobacteria and shows that the bifid shunt is the only pathway involved in energy recruitment from these two sugars. On the basis of our experimental results, a model of sugar metabolism in B. animalis subsp. lactis is proposed.

Project description:The complex formed between the enzyme triose phosphate isomerase (EC 5.3.1.1.), from rabbit and chicken muscle, and its substrate dihydroxyacetone phosphate was studied by 31P n.m.r. Two other enzyme-ligant complexes examined were those formed by glycerol 3-phosphate (a substrate analogue) and by 2-phosphoglycollate (potential transition-state analogue). Separate resonances were observed in the 31P n.m.r. spectrum for free and bound 2-phosphoglycollate, and this sets an upper limit to the rate constant for dissociation of the enzyme-inhibitor complex; the linewidth of the resonance assigned to the bound inhibitor provided further kinetic information. The position of this resonance did not vary with pH but remained close to that of the fully ionized form of the free 2-phosphoglycollate. It is the fully ionized form of this ligand that binds to the enzyme. The proton uptake that accompanies binding shows protonation of a group on the enzyme. On the basis of chemical and crystallographic information [Hartman (1971) Biochemistry 10, 146--154; Miller & Waley (1971) Biochem. J. 123, 163--170; De la Mare, Coulson, Knowles, Priddle & Offord )1972) Biochem. J. 129, 321--331; Phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc. Trans. 5, 642--647] this group is believed to be glutamate-165. On the other hand, the position of the resonance of D-glycerol 3 phosphate (sn-glycerol 1-phosphate) in the enzyme-ligand complex changes with pH, and both monoanion and dianon of the ligand bind, although dianion binds better. The substrate, dihydroxyacetone phosphate, behaves essentially like glycerol 3-phosphate. The experiments with dihydroxy-acetone phosphate and triose phosphate isomerase have to be carried out at 1 degree C because at 37 degrees C there is conversion into methyl glyoxal and orthophosphate. The mechanismof the enzymic reaction and the reasons for rate-enhancement are considered, and aspects of the pH-dependence are discussed in an Appendix.

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Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance.

Publications

Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance.

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