Defining the Structural Basis for Allosteric Product Release from E. coli Dihydrofolate Reductase Using NMR Relaxation Dispersion

Abstract

The rate-determining step in the catalytic cycle of E. coli dihydrofolate reductase is tetrahydrofolate (THF) product release, which can occur via an allosteric or an intrinsic pathway. The allosteric pathway, which becomes accessible when the reduced cofactor NADPH is bound, involves transient sampling of a higher energy conformational state, greatly increasing the product dissociation rate as compared to the intrinsic pathway that obtains when NADPH is absent. Although the kinetics of this process are known, the enzyme structure and the THF product conformation in the transiently formed excited state remain elusive. Here, we use side-chain proton NMR relaxation dispersion measurements, X-ray crystallography, and structure-based chemical shift predictions to explore the structural basis of allosteric product release. In the excited state of the E:THF:NADPH product release complex, the reduced nicotinamide ring of the cofactor transiently enters the active site where it displaces the pterin ring of the THF product. The p-aminobenzoyl-l-glutamate tail of THF remains weakly bound in a widened binding cleft. Thus, through transient entry of the nicotinamide ring into the active site, the NADPH cofactor remodels the enzyme structure and the conformation of the THF to form a weakly populated excited state that is poised for rapid product release.

Figure 1

(A) Schematic illustration of the allosteric pathway for product release from DHFR. The rate constants are for the L28F mutant. (B) Structure of the product tetrahydrofolate, with the pterin and benzoyl rings highlighted. (C) Structure of the reduced cofactor NADPH, highlighting the nicotinamide ring.

Figure 2

(A) Cartoon representation of L28F DHFR (blue) in complex with NADPH (green) superimposed on the structure of WT DHFR (red, PDB code 1RX1) in complex with NADPH (yellow). Both structures are in the closed state. Note that the NADPH molecules in the two structures are almost exactly superimposed and are difficult to distinguish in the figure. Side chains for the mutation site are shown in sticks, with the two alternative conformations observed for the F28 ring shown. (B) 2F_o – F_c map contoured at 2.0σ for NADPH in the L28F E:NADPH crystal structure.

Figure 3

(A) Methyl probes that undergo ¹H relaxation dispersion are represented by green spheres on the L28F E:ddTHF:NADP⁺ structure, which is an analogue for the L28F E:THF:NADPH structure. (B) Representative relaxation dispersion curves for a subset of proton methyl probes; 500 MHz data acquired with 16 scans per increment (red), 500 MHz data acquired with 32 scans per increment (green), and 800 MHz data acquired with 16 scans per increment (black). (C) Correlation plot for the dynamic chemical shift differences Δϖ versus the static chemical shift differences, Δδ = δ (E:THF:NADPH) – δ (E:NADPH). The dashed line indicates a slope = 1. Linear regression yields a slope = 1.09 and R² = 0.93, if I50Hδ¹ and L54Hδ² are excluded.

Figure 4

(A) Cartoon backbone representation of the L28F E:ddTHF:NADP⁺ crystal structure (PDB code 5CC9). NADP⁺ is omitted for clarity. The thickness of the loops is scaled by the B-value. The product analogue (ddTHF) is highlighted in yellow, residues that report on the conformation of the product in the excited state are shown as green sticks, and their specific methyl probes that undergo dispersion are shown as green spheres (A7 Hβ, I50 Hδ¹, and L54 Hδ²). (B) Overlay of the L28F E:NADPH (blue) and L28F E:ddTHF:NADP⁺(pink, PDB code 5CC9) crystal structures. The ligands are omitted for clarity. The thickness of the cartoon representation is scaled by the B-value. Helix C, which defines the upper edge of the product binding site, is shifted by 1.3 Å upward in the L28F E:NADPH structure relative to its position in the E:ddTHF:NADP⁺ structure. The structures shown in panels A and B are rotated by 90° with respect to each other. (C) Surface representation of DHFR in the crystal structure of the occluded L28F E:ddTHF:NADP⁺ complex showing the location of bound ddTHF (sticks and spheres, atom colors). The pterin ring is deeply buried in the active site (center left). (D) Model for the excited state of L28F E:THF:NADPH complex. The ddTHF is shown docked to the crystal structure of the closed L28F E:NADPH complex (gray surface). Rotation about the C10–C14 bond in the pABG tail rotates the pterin ring up and out of the active site to avoid steric clash with the nicotinamide ring of the NADPH (green).