Functionally important conformations of the Met20 loop in dihydrofolate reductase are populated by rapid thermal fluctuations

Abstract

Conformational changes in enzymes are well recognized to play an important role in the organization of the reactive groups for efficient catalysis. This study reveals atomic and energetic details of the conformational change process that precedes the catalytic reaction of the enzyme dihydrofolate reductase. The computed free energy profile provides insights into the ligand binding mechanism and a quantitative estimate of barrier heights separating different conformational states along the pathway. Studies show that the ternary complex comprised of NADPH cofactor and substrate dihydrofolate undergoes transitions between a closed state and an occluded state via an intermediate "open" conformation. During these transitions the largest conformational change occurs in the Met20 loop of DHFR and is accompanied by the motion of the cofactor into and out of the binding pocket. When the cofactor is out of the binding pocket, the enzyme frequently samples open and occluded conformations with a small (approximately 5 k(B)T) free energy barrier between the two states. However, when the cofactor is in the binding pocket, the closed conformation is thermodynamically most favored. The determination of a profile characterizing the position-dependent diffusion of the Met20 loop allowed us to apply reaction rate theory and deduce the kinetics of loop motions based on the computed free energy landscape.

Scheme 1

(a) Schematic representation of the catalytic pathway of DHFR as deduced from several kinetic and structural studies. In the holoenzyme E:NADPH and the Michaelis Menten complex E:NADPH:DHF, the Met20 loop adopts the closed conformation. In the three product complexes, E:NADP⁺, E:THF and E:NADPH:THF, the Met20 loop occurs in the occluded conformation. We have performed simulations capturing the enzyme’s transition between the closed and occluded state (step IIA, above), (b) Crystal structures of DHFR in the closed (left) and the occluded (right) states. In the closed state, the Met20 loop stacks against the nicotinamide ring of the cofactor while in the occluded state, the loop prevents cofactor from accessing the active site pocket.

Scheme 2

Free energy profile and structures indicating the conformational change of the Met20 loop in DHFR. (a) One-dimensional free energy profile along the ΔD_rmsd order parameter The error bars represent the standard deviation in the energy values determined from the set of three umbrella sampling MD simulations with different initial velocities. Inset depicts the enlarged view of the same plot, (b, c, & d) Representative structures, closed, open and occluded (top right to bottom right) corresponding to the three free energy minima (a, top left) along DHFR’s conformational change pathway.

Scheme 3

Characterization of the closed state. (a) View of the active site and Met20 loop corresponding to the closed state depicting atoms involved in the hydride transfer reaction. (b) The two-dimensional free energy surface along the ΔD_rmsd order parameter versus hydride transfer distance between the atom C6 of DHF and N5 of NADPH. In the closed state (ΔD_rmsd ~ −3.5 Å), the distance between the cofactor and substrate fluctuates in the range of 3–4 Å, suitable for promotion of the hydride transfer reaction.

Scheme 4

Free energy surface corresponding to the χ₁ dihedral angle of residues involved in organizing the active site of the enzyme for the subsequent catalytic reaction. (a) View of the active site showing the side chains of residues Ilel4 and Ile94 near the cofactor and the substrate, respectively, (b & c) Two-dimensional free energy surface corresponding to the χ₁ rotameric dihedral angle (N-C_α-C_β-C_γ1) along the reaction coordinate for residues Ilel4 (middle) and Ile94 (right). Both residues explore a minor trans conformation near the barrier region (see Scheme 2). In the trans rotameric state these residues interact unfavorably with the substrate and cofactor bringing the two reactants close to each other.

Scheme 5

Diffusion coefficients along the reaction coordinate. Mean values of the diffusion coefficients obtained from three independent umbrella sampling simulations is reported. Diffusion coefficients calculated using the harmonic oscillator approximation, $D=var(\mathrm{\Delta }{D}_{rmsd})/{\tau }_{\mathrm{\Delta }{D}_{rmsd}},$ from biased umbrella sampling simulations using a Langevin friction coefficient of 10 ps⁻¹.