Conformational change of the methionine 20 loop of Escherichia coli dihydrofolate reductase modulates pKa of the bound dihydrofolate

Abstract

We evaluate the pK(a) of dihydrofolate (H(2)F) at the N(5) position in three ternary complexes with Escherichia coli dihydrofolate reductase (ecDHFR), namely ecDHFR(NADP(+):H(2)F) in the closed form (1), and the Michaelis complexes ecDHFR(NADPH:H(2)F) in the closed (2) and occluded (3) forms, by performing free energy perturbation with molecular dynamics simulations (FEP/MD). Our simulations suggest that in the Michaelis complex the pK(a) is modulated by the Met20 loop fluctuations, providing the largest pK(a) shift in substates with a "tightly closed" loop conformation; in the "partially closed/open" substates, the pK(a) is similar to that in the occluded complex. Conducive to the protonation, tightly closing the Met20 loop enhances the interactions of the cofactor and the substrate with the Met20 side chain and aligns the nicotinamide ring of the cofactor coplanar with the pterin ring of the substrate. Overall, the present study favors the hypothesis that N(5) is protonated directly from solution and provides further insights into the mechanism of the substrate protonation.

Figure 1.

(A) The environment around the proton acceptor atom N⁵ of H₂F in the occluded and closed Michaelis complexes of ecDHFR. The pterin ring of H₂F and the nicotinamide portion of NADPH are displayed in solid ball-and-stick model. The Met20 loop is accentuated with a thick blue coil. Some of the residues that guard the N⁵ atom from water in the closed complex, namely 7, 18, 20, 22–25, 27, 28, 31, 49, and 52, are shown in the CPK model. The graphics were generated with Molscript and Raster3D (Kraulis 1991; Merritt and Bacon 1997). (B) Schematic representation of the active site and the hydride transfer step, along with the atomic labels used throughout the paper.

Figure 2.

Running cumulative ΔGs for the protonation of N⁵ of H₂F from the FEP/MD simulations in bulk water; in the observable ternary complex 1 ecDHFR(NADP⁺:H₂F); in the occluded Michaelis complex 3 ecDHFR(NADPH:H₂F); and in the closed Michaelis complex 2. Paired lines designate two independent simulations with different initial velocities.

Figure 3.

The distributions of the φ, ψ, and χ dihedral angles (see illustration [A] and text for description) that describe the substrate conformation. (B) Four sets of the distributions are shown in the order from bottom to top, namely, bulk water and the ternary complexes 1 through 3.

Figure 4.

The radial distribution functions (RDFs) between N⁵ of the substrate and oxygen of water molecules: (A) occluded Michaelis complex ecDHFR(NADPH:H₂F) 3; (B) closed Michaelis complex 2. Only the endpoints corresponding to the physical states of the substrate are displayed. The RDFs shown correspond to the simulations with identical initial conditions in all windows (see Supplemental materials for complete details).

Figure 5.

The distributions of the Met20 loop RMSD (see text for description) in (A) the closed complex ecDHFR(NADPH:H₂F) 2 and (B) the occluded complex 3. All FEP windows are shown (thin lines), along with the endpoint states (thick lines) to illustrate the overlap between the windows.

Figure 6.

The substrate pK_a in the closed complex ecDHFR(NADPH:H₂F) 2, as a function of the Met20 loop RMSD. The three curves refer to the results from the independent simulations 1 (squares) and 2 (triangles) with different initial velocities and the combined (crosses) result.

Figure 7.

The interaction of the Met20 side chain with the substrate and cofactor in the closed complex. (A) An illustration of the relative position of the Met20 side chain, substrate, and cofactor in the closed complex ecDHFR(NADPH:H₂F) 2. The distributions of the (Met20)S–N⁵(substrate) distances in complex 2. (B) The overall normalized distributions. (C) The renormalized distributions obtained after filtering configurations by Met20 loop RMSD <1.0 Å. In B and C only the distributions from the simulation windows that correspond to the physical states of the substrate are displayed.

Figure 8.

Cofactor orientation in the closed complex. (A) The two cofactor conformations observed under the closed Met20 loop. The distributions for the angle between the mean planes formed by pterin and nicotinamide rings of the substrate and cofactor, respectively. (B) The overall normalized distributions. (C) The renormalized distributions obtained after filtering configurations by Met20 loop RMSD <1.0 Å. In B and C, only the distributions from the simulation windows that correspond to the physical states of the substrate are displayed.

Figure 9.

The kinetic schemes involving enzyme and substrate protonation and their effect on the hydride transfer rate. (A) The two schemes proposed. (B) The resulting hydride transfer kinetic profiles as a function of pH. See text for the parameters used to obtain the profiles. Note that contributions to the hydride transfer rate from both channels are shown for Scheme II, along with the cumulative rate.