title: Structure and dynamics of the G121V dihydrofolate reductase mutant: lessons from a transition-state inhibitor complex.
abstract: It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on (15)N and (2)H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in µs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect.
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Randall V
authors.initials: RV
authors.lastname: Mauldin
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Sapienza
authors.forename: Chad M
authors.initials: CM
authors.lastname: Petit
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Lee
mesh_terms: D016880:Anisotropy; D008727:Methotrexate / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense / Q000235:genetics*; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
chemical_list: D009587:Nitrogen Isotopes; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate