Connecting protein conformational dynamics with catalytic function as illustrated in dihydrofolate reductase

Abstract

Combined quantum mechanics/molecular mechanics molecular dynamics simulations reveal that the M20 loop conformational dynamics of dihydrofolate reductase (DHFR) is severely restricted at the transition state of the hydride transfer as a result of the M42W/G121V double mutation. Consequently, the double-mutant enzyme has a reduced entropy of activation, i.e., increased entropic barrier, and altered temperature dependence of kinetic isotope effects in comparison with those of wild-type DHFR. Interestingly, in both wild-type DHFR and the double mutant, the average donor-acceptor distances are essentially the same in the Michaelis complex state (~3.5 Å) and the transition state (2.7 Å). It was found that an additional hydrogen bond is formed to stabilize the M20 loop in the closed conformation in the M42W/G121V double mutant. The computational results reflect a similar aim designed to knock out precisely the dynamic flexibility of the M20 loop in a different double mutant, N23PP/S148A.

Figure 1

X-ray structure of the ternary complex between E. coli dihydrofolate reductase, folate substrate and NADP⁺ cofactor (PDB code: 1RX2). Secondary structure and loop nomenclature are indicated.

Figure 2

Computed classical mechanical potential of mean force for the hydride transfer in wild-type DHFR (a) and in the double mutant M42W/G121V at 5 °C, 25 °C, and 45 °C. The reaction coordinate is defined as the difference in the distance of the transferring hydrogen (H4) from the donor carbon (C4N) of the cofactor and the acceptor carbon (C6) of the substrate.

Figure 2

Computed classical mechanical potential of mean force for the hydride transfer in wild-type DHFR (a) and in the double mutant M42W/G121V at 5 °C, 25 °C, and 45 °C. The reaction coordinate is defined as the difference in the distance of the transferring hydrogen (H4) from the donor carbon (C4N) of the cofactor and the acceptor carbon (C6) of the substrate.

Figure 3

Eyring plots for the hydride transfer in wild-type DHFR (red) and in M42W/G121V mutant (black). Rate constants are determined using quantum transition state theory in which nuclear quantum effects are included in the estimated free energy barrier.

Figure 4

Computed average vibrational free energies for H-transfer (blue) and D-transfer (green) in wild-type DHFR. The results are fitted to an inverse Eckart function, and the location of the transition state at 5 °C and 45 °C, determined after the vibrational free energies are included in the classical mechanical potential of mean force, are indicated by the vertical lines. The Boltzmann factor of between H- and D-transfers gives the vibrational free energy contribution (dominantly zero-point effects) to the overall KIE.

Figure 5

Comparison of the Arrhenius plots of experimental and computational intrinsic H/D kinetic isotope effects in wild-type and M42W/G121V mutant DHFR.

Figure 6

Comparison of snap shot structures in the reactant state (Michaelis complex) in blue and in the transition state (brown) for the hydride transfer in wild-type DHFR (a) and in the M42W/G121V double mutant (b).

Figure 7

Difference is mean square fluctuations (MSF) of backbone Cα atoms between wild-type DHFR and the M42W/G121V double mutant, MSF[DM]-MSF[WT], in the Michaelis complex reactant state (black) and in the transition state (red). Negative values indicate regions with greater structural fluctuations in the WT enzyme, whereas positive regions reflect enhanced fluctuations in the DM enzyme.

Figure 8

Illustration of regional variations between wild-type DHFR and the M42W/G121V double mutant in going from the reactant state (Michaelis complex) to the transition state. This corresponds to the difference of the two MSFs shown in Figure 7. Regions showing reduced fluctuations to reach the transition state in the double mutant relative to that in the WT enzyme are given in red, while enhanced fluctuations are in blue (almost nonexistent except the C-D loop region on the top).

Figure 9

Illustration of the dynamic motions of the lowest frequency quasiharmonic mode for the wild-type (WT) and M42W/G121V double mutant (DM) in the reactant state (RS) and the transition state (TS). A sequence of six structures stretched up to 2σ from the mean are superimposed.

Figure 10

Structures highlighting hydrogen bonding interactions between Q18 of the M20 loop and the cofactor sugar and amino acids (H45 and S49) of the αC helix. M42 in the wild-type DHFR and W42 in the double mutant are shown as stick models in the back of the αC helix.

Scheme 1

Schematic representation of atoms treated explicitly by electronic structure theory and by molecular mechanics for the NADPH cofactor (top) and 7,8-dihydrofolate substrate (bottom). The symbol B indicates a boundary atom which is treated both as a QM and an MM atom in the generalized hybrid orbital (GHO) method. Adapted from ref. (30) with permission from the American Chemical Society.

Scheme 2

A schematic description of extreme models using protein reorganization energy. In model (a), the preorganization effect is disrupted due to distant mutantions along with increased donor-acceptor distance (not shown in the energy gap coordinate). This results in a greater reorganization energy, i.e., free energy barrier, to reach the transition state at the diabatic crossing point. In model (b), the donor and acceptor species have the same distance and preorganization effect in the Michaelis complex both in the wild-type and double mutant enzymes, but remote mutations affect protein conformational flexibility towards the transition state, which is reported by increased force constant for the protein response in the diabatic picture.