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

Xiaorong Yang, Jesse L. Welch, Jamie Jon Arnold, David D. Boehr

Research output: Contribution to journalArticle

37 Citations (Scopus)

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- 13C]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- 13C]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.

Original languageEnglish (US)
Pages (from-to)9361-9371
Number of pages11
JournalBiochemistry
Volume49
Issue number43
DOIs
StatePublished - Nov 2 2010

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RNA Replicase
Poliovirus
Methionine
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Mutation
Nucleotides
RNA
Catalysis
Chemical shift
Ligands
Viruses
Relaxation time
Substitution reactions
Enzymes
Proteins
methionine methyl ester

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "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- 13C]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- 13C]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 {\AA} 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.",
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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; Welch, Jesse L.; Arnold, Jamie Jon; Boehr, David D.

In: Biochemistry, Vol. 49, No. 43, 02.11.2010, p. 9361-9371.

Research output: Contribution to journalArticle

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T1 - Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance

AU - Yang, Xiaorong

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AU - Boehr, David D.

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N2 - 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- 13C]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- 13C]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.

AB - 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- 13C]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- 13C]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.

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