Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands

Abstract

Enzyme catalysis can be described as progress over a multi-dimensional energy landscape where ensembles of interconverting conformational substates channel the enzyme through its catalytic cycle. We applied NMR relaxation dispersion to investigate the role of bound ligands in modulating the dynamics and energy landscape of Escherichia coli dihydrofolate reductase to obtain insights into the mechanism by which the enzyme efficiently samples functional conformations as it traverses its reaction pathway. Although the structural differences between the occluded substrate binary complexes and product ternary complexes are very small, there are substantial differences in protein dynamics. Backbone fluctuations on the micros-ms timescale in the cofactor binding cleft are similar for the substrate and product binary complexes, but fluctuations on this timescale in the active site loops are observed only for complexes with substrate or substrate analog and are not observed for the binary product complex. The dynamics in the substrate and product binary complexes are governed by quite different kinetic and thermodynamic parameters. Analogous dynamic differences in the E:THF:NADPH and E:THF:NADP(+) product ternary complexes are difficult to rationalize from ground-state structures. For both of these complexes, the nicotinamide ring resides outside the active site pocket in the ground state. However, they differ in the structure, energetics, and dynamics of accessible higher energy substates where the nicotinamide ring transiently occupies the active site. Overall, our results suggest that dynamics in dihydrofolate reductase are exquisitely "tuned" for every intermediate in the catalytic cycle; structural fluctuations efficiently channel the enzyme through functionally relevant conformational space.

Fig. 1.

Structural comparison of E.coli DHFR substrates, products and complexes. (A). The hydride transfer reaction, indicating the chemical structures of folate (FOL), dihydrofolate (DHF) and tetrahydrofolate (THF). (B). Superposition of the backbone structures of the E:FOL (PDB 1rx7, Coral), E:ddTHF (5,10-dideazatetrahydrofolate; PDB 1rx5, Blue), and E:ddTHF:NADPH (PDB 1rx6, Yellow) complexes of E. coli DHFR. The Met20 loop in all three complexes is in the occluded conformation. The folate, ddTHF, and NADPH are indicated with Spheres; Pink for folate, Green for ddTHF, and Gray for NADPH. In the occluded conformation, the Met20 loop protrudes into the nicotinamide binding pocket; consequently, the nicotinamide-ribose moiety of NADPH projects into the solvent in the E:ddTHF:NADPH complex and the nicotinamide ring is disordered (10).

Fig. 2.

Dynamics in E.coli DHFR binary complexes. The location of residues displaying ¹⁵N R₂ relaxation dispersion is shown on the left for (A) E:FOL, (B) E:DHF, and (C) E:THF complexes. The backbone nitrogen atoms of residues displaying R₂ dispersion are shown as colored spheres (active site loops, Red; cofactor binding cleft, Green; substrate/product binding pocket, Gold; other active site residues, Gray; and C-terminal associated region, Blue). The figure was prepared by using MOLMOL (42). Representative R₂ relaxation dispersion curves at 800 MHz for N23 (Red) and S63 (Green) are shown on the right. A representative set of R₂ relaxation dispersion data at 300 K is shown in Figs. S4 and S5.

Fig. 3.

Energy landscapes of the binary complexes. Temperature dependence of the exchange kinetics in (A) the active site region and (B) the C-terminal associated region for E:FOL (Red Triangles), E:DHF (Blue Circles) and E:THF (Green Squares). (C) Comparison of thermodynamics for the E:FOL and E:THF complexes at 300 K. Thermodynamic barriers were calculated by using transition-state theory (SI Text). ΔG, ΔH, and TΔS traces are colored green, blue, and red, resp. The ground-state conformation for E:FOL and E:THF are used as reference states (G = 0 kcal/mol).

Fig. 4.

Dynamics in the product ternary complexes E:THF:NADP⁺ and E:THF:NADPH. The backbone nitrogen atoms of residues displaying ¹⁵N R₂ relaxation dispersion for (A) E:THF:NADP⁺ and (B) E:THF:NADPH are indicated as colored spheres (same color scheme as in Fig. 2). This figure was prepared by using MOLMOL (42) from relaxation dispersion data reported previously [Supplemental Online Material for (12)]. (C) Energy level diagram for the E:FOL:NADP⁺, E:THF:NADP⁺, and E:THF:NADPH complexes based on thermodynamic data in (24). The occluded conformation with the nicotinamide ring outside of the active site pocket is taken as a common reference (G = 0 kcal/mol).

Fig. 5.

Quantitative free energy changes in the catalytic cycle of E.coli DHFR. The apo-enzyme is taken as the reference state (G = 0 kcal/mol). The free energy landscape changes in response to ligand binding and release. The ground state free energies are calculated based on previously published equilibrium and kinetic binding data (36). Arrows suggest a path through conformational space.