A distal mutation perturbs dynamic amino acid networks in dihydrofolate reductase

Abstract

Correlated networks of amino acids have been proposed to play a fundamental role in allostery and enzyme catalysis. These networks of amino acids can be traced from surface-exposed residues all the way into the active site, and disruption of these networks can decrease enzyme activity. Substitution of the distal Gly121 residue in Escherichia coli dihydrofolate reductase results in an up to 200-fold decrease in the hydride transfer rate despite the fact that the residue is located 15 Å from the active-site center. In this study, nuclear magnetic resonance relaxation experiments are used to demonstrate that dynamics on the picosecond to nanosecond and microsecond to millisecond time scales are changed significantly in the G121V mutant of dihydrofolate reductase. In particular, picosecond to nanosecond time scale dynamics are decreased in the FG loop (containing the mutated residue at position 121) and the neighboring active-site loop (the Met20 loop) in the mutant compared to those of the wild-type enzyme, suggesting that these loops are dynamically coupled. Changes in methyl order parameters reveal a pathway by which dynamic perturbations can be propagated more than 25 Å across the protein from the site of mutation. All of the enzyme complexes, including the model Michaelis complex with folate and nicotinamide adenine dinucleotide phosphate bound, assume an occluded ground-state conformation, and we do not observe sampling of a higher-energy closed conformation by (15)N R2 relaxation dispersion experiments. This is highly significant, because it is only in the closed conformation that the cofactor and substrate reactive centers are positioned for reaction. The mutation also impairs microsecond to millisecond time scale fluctuations that have been implicated in the release of product from the wild-type enzyme. Our results are consistent with an important role for Gly121 in controlling protein dynamics critical for enzyme function and further validate the dynamic energy landscape hypothesis of enzyme catalysis.

Figure 1

Structures representing the (A) occluded (PDB 1RX7)²⁵ and (B) closed loop conformations (PDB 1RX2)²⁵ of E. coli DHFR. The active site loops are colored in green (Met20), blue (F–G), and red (G–H). Residues involved in hydrogen-bonding interactions between these loops are shown as sticks and labeled. Hydrogen bonds that stabilize the different loop conformations are indicated with dashed lines. In the occluded conformation of the Met20 loop (A), hydrogen bonds are formed between Asn23 in the F–G loop and Ser148 in the Met20 loop. In the closed conformation of the Met20 loop (B), hydrogen bonds are formed between Asp122 in the F–G loop and Gly15 and Glu17 in the Met20 loop. The figure was prepared using PyMOL.

Figure 2

Backbone amide ¹H^N and ¹⁵N chemical shift differences between WT and G121V DHFR complexes, (A) E:FOL, (B) E:FOL:NADP⁺, (C) E:THF:NADP⁺ and (D) E:THF:NADPH. The weighted average shift difference (Δδ_ave) for each residue was calculated as ((Δδ_H)² + (Δδ_N/5)²)^1/2 where Δδ_H,N is δ(G121V) – δ(WT). For E:FOL:NADP⁺ (B), the black line is a comparison between G121V E:FOL:NADP⁺ and WT E:FOL:NADP⁺ and the red line is a comparison between G121V E:FOL:NADP⁺ and G121V E:THF:NADP⁺.

Figure 3

Backbone amide and tryptophan imino model-free parameters S², τ_e and R_ex for the WT (black) and G121V (red) E:FOL complexes extracted from fits to data at 500 and 600 MHz. Values for R_ex are reported for 600 MHz.

Figure 4

Changes in dynamics in the G121V E:FOL complex, mapped onto the DHFR structure (PDB 1RX7)²⁵ A. Change in backbone amide S² between the WT and G121V E:FOL binary complexes. Blue, increase in S² showing restriction of ps-ns backbone motion in the mutant; red, decreased S², indicating increased amplitude of backbone motion in mutant. B. Location of residues that show changes in amplitude (S²) and/or timescale (τ_e) of ps-ns backbone amide motions caused by the G121V mutation.

Figure 5

Model-free parameters derived from methyl-incorporated deuterium relaxation data for the E:FOL complex of G121V DHFR. Shown are the methyl axis order parameters (S²_axis) and internal correlation times (τ_e) for A the G121V E:FOL complex and B the deviations from WT behavior. Differences are shown as G121V minus WT. Model-free parameters were extracted from fits to R₁ (²H) and R_1ρ (²H) at ¹H spectrometer frequencies of 600 and 800 MHz. Model-free parameters for the WT complex are from published work. Parameters for Met20-ε and Met42-ε in WT DHFR were derived from 600 MHz (¹H frequency) spectra of a binary complex with dihydrofolate, used to resolve overlap in the spectra of the E:FOL complex. Changes in dynamics for Met20 and Met42 are indicated with a star in B. Ala-β, Thr-γ2, Met-ε, Ile-γ2, Leu-δ1 and Val-γ1 are shown as open squares. Ile-δ1, Leu-δ2 and Val-γ2 are shown as filled squares.

Figure 6

Location of methyl groups that show altered S²_axis. Blue, increased motional restriction (decreased amplitude/increased S²_axis), red, increased motion (decreased S²_axis), green, methyl groups that show slower time scale motion (larger τ_e) in the G121V mutant. Tryptophan side chains that show changes in Nε order parameters or τ_e are shown. Red Nε/pink side chain indicates increased flexibility, blue Ne/pale blue side chain indicates more restricted side chain dynamics in the mutant protein. The mutation site, Gly121, is indicated and folate is shown as yellow sticks. Selected sites in the backbone and side-chain are labeled. Figures were prepared using PyMol.

Figure 7

Comparison of μs-ms timescale backbone dynamics between WT (left) and G121V (right) DHFR complexes (A) E:FOL, (B) E:FOL:NADP⁺, (C) E:THF:NADP⁺ and (D) E:THF:NADPH. Residues displaying conformational exchange (R_ex) are highlighted as colored spheres (red – Met20, FG and GH active-site loops, green – cofactor binding cleft, gold – substrate/product binding site, grey – other residues, pale blue (at the back of each structure) – C-terminal associated region). The coordinates used are 1RX7 (WT and G121V E:FOL), 3QL3/1RX2 (WT E:FOL:NADP⁺, a closed conformation). For everything else the coordinate set 1RX6 (the 5, 10 dideazatetrahydrofolate (ddTHF)-NADPH complex) was used to model an occluded conformation with the adenosine ring bound but with the nicotinamide disordered and solvent-exposed outside the active site. Substrate molecules (ddTHF, FOL) are shown as yellow sticks, cofactors (NADP⁺, NADPH are shown as green and red (phosphate) sticks).

Figure 8

A. Temperature dependence of the conformational exchange kinetics in the G121V E:FOL:NADP⁺ complex for the active-site region. Rate constants for the transitions from the excited state to the ground state [k_BA (■)] and from the ground state to the excited state [k_AB (▲)] and the equilibrium constant [k_BA/k_AB (●)] are plotted. B. Thermodynamic comparison of the G121V E:FOL:NADP⁺ complex (occluded ground state, unknown excited state; o → ?) with WT E:FOL:NADP⁺ (closed ground state, occluded excited state; c → o) and WT E:THF:NADP⁺ (occluded ground state, closed excited state; o → c) dynamics at 298 K. Thermodynamic barriers were calculated using transition-state theory according to Materials and Methods. ΔG, ΔH and TΔS traces are colored green, blue and red respectively.