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 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.

Protein motions and dynamic effects in enzyme catalysis

“…Indeed, the G123V mutation in MpDHFR has little effect on the single turnover rate constant or its observed KIE at pH 7, consistent with the proposal that interactions between the FG and M20 loops do not play a significant role in MpDHFR catalysis. 49 Binding of NADPH and the inhibitor methotrexate to EcDHFR-G121V forms a putative mimic of the transition state and causes the enzyme to adopt a closed conformation, 26 confirming that EcDHFR-G121V is capable of forming this conformation for hydride transfer. However, the mutation leads to aberrant millisecond conformational switching of the M20 and FG loops, 26 as would be expected when the closed conformation is strongly destabilised, suggesting that these motions are anti-catalytic and destabilise the optimum active site configuration.…”

“…49 Binding of NADPH and the inhibitor methotrexate to EcDHFR-G121V forms a putative mimic of the transition state and causes the enzyme to adopt a closed conformation, 26 confirming that EcDHFR-G121V is capable of forming this conformation for hydride transfer. However, the mutation leads to aberrant millisecond conformational switching of the M20 and FG loops, 26 as would be expected when the closed conformation is strongly destabilised, suggesting that these motions are anti-catalytic and destabilise the optimum active site configuration. 26 The EcDHFR-M42W complex with NADPH and methotrexate also forms a closed conformation and shows slower millisecond motion than the wild-type enzyme.…”

Loop Interactions during Catalysis by Dihydrofolate Reductase from Moritella profunda

“…18 On the other hand, the transition state of EcDHFR-G121V, modeled by the E·NADPH·MTX complex for NMR and X-ray studies, 3,21 has been shown to form a closed complex (as is necessary for catalysis to occur) but with significant excursions of the FG and M20 loops away from the accurate alignment required for reaction. 21 This is in agreement with previous computational studies, which showed that the G121V mutation leads to an altered transition state structure in EcDHFR, with a concomitant increase in the activation free energy barrier. 17,20 These results suggest that EcDHFR-G121V is capable of adopting the closed conformation but that the conformational fluctuations are different from those of the wild-type enzyme.…”

“…When the closed conformation is destabilized by mutations in the FG loop, the occluded conformation becomes the ground state of EcDHFR in the Michaelis complex. Reaction can then only occur from a minor population of the overall ensemble, 21 as NADPH and DHF are held in positions unsuitable for reaction in the occluded conformation. 3 As MpDHFR lacks the key residue that stabilizes the occluded conformation (DOI: 10.1021/bi500507v), loss of hydrogen bonds that stabilize the closed conformation in MpDHFR will simply lead to formation of a destabilized closed conformation rather than favoring an occluded conformation.…”

Controlling Active Site Loop Dynamics in the (β/α)8 Barrel Enzyme Indole-3-Glycerol Phosphate Synthase

“…A similar example is found in the E. coli DHFR enzyme, in which the FG and GH loops compete for interactions with the active-site Met20 loop [49]. Amino acid substitutions in either the FG and GH loops that disrupt these interactions lead to decreases in catalytic efficiency [50][51][52] and changes to the µs-ms timescale Met20 loop dynamics [53][54][55].…”