title: A distal mutation perturbs dynamic amino acid networks in dihydrofolate reductase.
abstract: Correlated networks of amino acids have been proposed to play a fundamental role in allostery and enzyme catalysis. These networks of amino acids can be traced from surface-exposed residues all the way into the active site, and disruption of these networks can decrease enzyme activity. Substitution of the distal Gly121 residue in Escherichia coli dihydrofolate reductase results in an up to 200-fold decrease in the hydride transfer rate despite the fact that the residue is located 15 Å from the active-site center. In this study, nuclear magnetic resonance relaxation experiments are used to demonstrate that dynamics on the picosecond to nanosecond and microsecond to millisecond time scales are changed significantly in the G121V mutant of dihydrofolate reductase. In particular, picosecond to nanosecond time scale dynamics are decreased in the FG loop (containing the mutated residue at position 121) and the neighboring active-site loop (the Met20 loop) in the mutant compared to those of the wild-type enzyme, suggesting that these loops are dynamically coupled. Changes in methyl order parameters reveal a pathway by which dynamic perturbations can be propagated more than 25 Å across the protein from the site of mutation. All of the enzyme complexes, including the model Michaelis complex with folate and nicotinamide adenine dinucleotide phosphate bound, assume an occluded ground-state conformation, and we do not observe sampling of a higher-energy closed conformation by (15)N R2 relaxation dispersion experiments. This is highly significant, because it is only in the closed conformation that the cofactor and substrate reactive centers are positioned for reaction. The mutation also impairs microsecond to millisecond time scale fluctuations that have been implicated in the release of product from the wild-type enzyme. Our results are consistent with an important role for Gly121 in controlling protein dynamics critical for enzyme function and further validate the dynamic energy landscape hypothesis of enzyme catalysis.
authors.affiliation: Department of Integrative Structural and Computational Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: David D
authors.initials: DD
authors.lastname: Boehr
authors.affiliation: Department of Integrative Structural and Computational Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Schnell
authors.affiliation: Department of Integrative Structural and Computational Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: McElheny
authors.affiliation: Department of Integrative Structural and Computational Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Sung-Hun
authors.initials: SH
authors.lastname: Bae
authors.affiliation: Department of Integrative Structural and Computational Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Brendan M
authors.initials: BM
authors.lastname: Duggan
authors.affiliation: The Pennsylvania State University, Department of Chemistry, 414 Wartik Laboratory, University Park, PA 16802, USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
authors.affiliation: Department of Integrative Structural and Computational Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: H Jane
authors.initials: HJ
authors.lastname: Dyson
authors.affiliation: Department of Integrative Structural and Computational Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Wright
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics
chemical_list: D000596:Amino Acids; D013762:Tetrahydrofolate Dehydrogenase