NMR study of the role of M42 in the solution dynamics of E. COLI dihydrofolate reductase

It is widely recognized that key positions throughout a protein’s structure contribute unequally to function. In light of recent studies that suggest protein dynamics are required for function, a number of these residues may serve to promote motions required for ligand binding and catalysis. In the present NMR study, the conformational dynamics of the dihydrofolate reductase (DHFR) mutant M42W, in the presence of methotrexate and NADPH, are characterized and compared to the wild-type enzyme. M42 is distal to the active site, yet the M42W substitution regulates catalysis and ligand affinity, and is therefore analogous to an allosteric modulator of DHFR function. To gain understanding of how this mutation regulates activity, we employ a “pandynamic” strategy by measuring conformational fluctuations of backbone amide and side-chain methyl groups on multiple timescales. Changes in ps-ns dynamics indicate that the mutational effects are propagated throughout a network of interacting residues within DHFR, consistent with a role for M42 as a dynamic communication hub. On the μs-ms timescale, mutation increases the rate of switching in the catalytic core. Mutation also introduces switching in the adenosine binding subdomain that occurs at a higher frequency than in the catalytic core, and which correlates with the rate of product release for M42W-DHFR. Finally, a structurally inferred analysis of side-chain dynamics suggests that the M42W mutation dampens motional contributions from non-local effects. These data show that the M42W mutation alters the dynamics of DHFR and are consistent with theoretical analysis that suggests the mutation disrupts motion that promotes catalysis

Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State

The arduous task of rationally designing small molecule enzyme inhibitors is complicated by the inherent flexibility of the protein scaffold. To gain insight into the changes in dynamics associated with small molecule based inhibition, we have characterized, using NMR spectroscopy, E. coli dihydrofolate reductase in complex with two drugs: methotrexate and trimethoprim. The complexes allowed the intrinsic dynamic effects of drug binding to be revealed within the context of the “closed” structural ensemble. Binding of both drugs results in an identical decoupling of global motion on the micro- to millisecond timescale. Consistent with a change in overall dynamic character, the drugs’ perturbations to pico- to nanosecond backbone and side-chain methyl dynamics are also highly similar. These data show that the inhibitors simultaneously modulate slow concerted switching and fast motions at distal regions of DHFR, providing a dynamic link between the substrate binding site and distal loop residues known to affect catalysis

Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes, and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times, or koff, should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into koff for a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase that vary by six orders of magnitude in binding affinity. NMR relaxation dispersion experiments revealed a common set of residues near the binding site that undergo a concerted, millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with Ki and koff, suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although kconf,forward is faster than koff, use of the ligand series allowed for connections to be drawn between kinetic events on different timescales

Structure and Dynamics of the G121V Dihydrofolate Reductase Mutant: Lessons from a Transition-State Inhibitor Complex

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 15N and 2H 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

G121V dependent changes in backbone and side-chain dynamics on the ps-ns timescale.

<p>Changes in backbone <i>S</i>² (A) and τ_e (B) order parameters determined from ¹⁵N relaxation experiments. Changes in side-chain <i>S</i>²_axis (D) and τ_e,_axis (E) order parameters determined from ²H relaxation experiments. Plotted values were obtained by subtracting mutant (E^G121V:NADPH:MTX) from wild-type (E:NADPH:MTX) parameters with significant changes (>1.5σ) highlighted in red (backbone) and blue (side-chain). Significant changes are also mapped onto the structure using red spheres for backbone (C) and blue spheres for side-chain (F). Greens spheres denote residues that have significant changes in both backbone and side-chain order parameters. An area of 5 Å around the active site is highlighted in green.</p

Model-free analysis of tryptophan indole ¹⁵N relaxation within E:NADPH:MTX and E^G121V:NADPH:MTX complexes.

a<p><i>R</i>_ex values are based on model-free fits.</p

DHFR subdomain and loop nomenclature.

<p>Structure of DHFR complexed with NADPH (purple) and methotrexate (yellow) (pdbid 1RX3). Adenosine binding domain is shown in light blue while the loops domain is shown in wheat. The Met20 (red), F–G (blue), G–H (green) loops are labeled. The site of mutation (G121V) is indicated by a cyan sphere.</p

Methotrexate binding traps G121V in the closed conformation.

<p>(A) ¹H–¹⁵N HSQC spectra of E:NADPH:MTX (black) overlain with the ¹H–¹⁵N HSQC spectra of E^G121V:NADPH:MTX (red). (B) The reduced change in chemical shift is calculated using the following formula and is subsequently plotted as a function of residue number: . The changes as a result of mutation (G121V vs. WT) are shown in black and a bona fide closed–occluded change are plotted in red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033252#pone.0033252-Osborne1" target="_blank">[23]</a>. The plot shows that the pattern of changes as a result of mutation can be attributed to differences in the local chemical environment and not large-scale structural change. The Met20 (residues 9–23), F–G (residues 117–131), and G–H (residues 142–149) loops are highlighted.</p

Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes, and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times, or k(off), should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) for a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase that vary by six orders of magnitude in binding affinity. NMR relaxation dispersion experiments revealed a common set of residues near the binding site that undergo a concerted, millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with K(i) and k(off), suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although k(conf,forward) is faster than k(off), use of the ligand series allowed for connections to be drawn between kinetic events on different timescales