Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis

Abstract

Dynamic processes are implicit in the catalytic function of all enzymes. To obtain insights into the relationship between the dynamics and thermodynamics of protein fluctuations and catalysis, we have measured millisecond time scale motions in the enzyme dihydrofolate reductase using NMR relaxation methods. Studies of a ternary complex formed from the substrate analog folate and oxidized NADP+ cofactor revealed conformational exchange between a ground state, in which the active site loops adopt a closed conformation, and a weakly populated (4.2% at 30 degrees C) excited state with the loops in the occluded conformation. Fluctuations between these states, which involve motions of the nicotinamide ring of the cofactor into and out of the active site, occur on a time scale that is directly relevant to the structural transitions involved in progression through the catalytic cycle.

Fig. 1.

Schematic representation of the catalytic cycle of E. coli DHFR for steady-state turnover under saturating substrate conditions (9). The active site loops in the holoenzyme (E^NH) and Michaelis complex () are in the closed conformation. The ternary () and binary (E_THF) product complexes and the product release complex () all adopt the occluded conformation (10, 11). NH and N⁺ refer to reduced and oxidized cofactor, NADPH and NADP⁺, respectively.

Fig. 2.

Relaxation dispersion measurements for the ternary complex of DHFR with folate and NADP⁺.(a) ¹⁵N relaxation dispersion profiles for representative residues from the Met-20 (Ala-19, blue lines and symbols), FG (Glu-120, green), and GH (His-149, red) loops. The data were acquired at 306.4 K at spectrometer frequencies of 500 (open symbols) and 800 (filled symbols) MHz. The solid lines show the fits to the general exchange equation (Eq. 2) with k_ex = 555 s^–1 and p_b = 5.5%. (b) Structure of the ternary complex of E. coli DHFR with bound folate and NADP⁺ (10) showing the location of residues that exhibit exchange broadening. Residues that exhibit ¹⁵N relaxation dispersion due to conformational fluctuations with k_ex = 555 s^–1 (at 306.4 K) are colored orange; residues fit to k_ex = 1,280 s^–1 are colored red. Residues that exhibit severe exchange broadening at all temperatures are colored brown, and those that show no dispersive behavior are colored blue. Proline and residues with missing or overlapped ¹⁵N resonances are white. The bound NADP⁺ is pink and folate is shown in green.

Fig. 3.

Correlation of chemical shift differences observed between closed and occluded ternary complexes of DHFR and the chemical shift differences determined from the relaxation dispersion measurements. (a) Difference in ¹⁵N chemical shift (Δδ) between the closed DHFR:folate:NADP⁺ complex and the occluded DHFR:folate:DHNADPH complex (28). (b) Scatter plot showing correlation (R = 0.99) between Δω, determined from ¹⁵N relaxation dispersion curves fit with k_ex = 555 s^–1 (at 306.4 K), and the equilibrium ¹⁵N chemical shift difference, Δδ. Residues for which the sign of Δω can be determined experimentally (see text) are indicate by filled circles. Absolute values of both Δω and Δδ are plotted (open circles) when the sign of Δω is unknown. The line of best fit is shown (slope = 1.12 ± 0.05). Residues for which Δδ is <0.4 ppm, and which are therefore relatively insensitive to the closed–occluded conformational transition, are omitted from the correlation plot.

Fig. 4.

Determination of thermodynamic parameters for the closed–occluded exchange process. Relaxation dispersion profiles for Glu-118 (a) and His-149 (b) at 303.4 (blue), 306.4 (green), 309.4 (orange), and 312.4 (red) K. The data were acquired at spectrometer frequencies of 500 (open symbols) and 800 (filled symbols) MHz. The solid lines show the fits to the general exchange equation (Eq. 2). (c) Arrhenius and van't Hoff plots for the forward (k_f) and backward (k_b) rate constants and the equilibrium constant (K_eq = k_b/k_f) obtained from global fits of k_ex and p_b for the exchange process affecting the active site loops. (d) Schematic energy level diagram showing enthalpy (H), free energy (G), and entropy (TS) at 298 K of the occluded excited state and the transition state relative to the closed ground state.

Fig. 5.

Structural changes associated with the closed-to-occluded transition. The polypeptide backbone surrounding the active site and the adenosine binding site is shown as a thick tube, with the central region of the Met-20 loop emphasized in darker colors. The substrate and cofactor are colored yellow and pink, respectively. The closed conformation is represented by the x-ray structure of the DHFR:folate:NADP⁺ complex (PDB ID code 1RX2) (10). The nicotinamide ring occupies the active site, with the Met-20 loop packed against it. The occluded conformer is represented by the structure of the ternary complex of DHFR with 5,10-dideazatetrahydrofolate and NADPH (PDB ID code 1RX6) (10); the nicotinamide-ribose moiety is absent from the crystal structure and has been modeled with the nicotinamide ring projecting into solution. In the occluded conformation, the Met-20 loop blocks the nicotinamide-ribose binding pocket.

Fig. 6.

Schematic diagram showing the additional steps (in boxes) implicated in the transitions from the closed Michaelis complex to the occluded product ternary complex. The experimentally observed kinetic intermediates (9) are enclosed in circles, and closed and occluded conformations are indicated in red and blue, respectively. Schematic free energy diagrams indicating the relative energies of the closed and occluded states, deduced from the present relaxation dispersion studies of the DHFR:folate:NADP⁺ model of the Michaelis complex, are shown. Excited states associated with the closed–occluded equilibria are marked with an asterisk. “IN” and “OUT” denote whether the nicotinamide ring of the cofactor occupies the active site or is displaced by the Met-20 loop. The estimated rates, extrapolated to 25°C, for the conformational transitions are indicated.