Computational studies of the farnesyltransferase ternary complex part II: the conformational activation of farnesyldiphosphate

Abstract

Studies aimed at elucidating the reaction mechanism of farnesyltransferase (FTase), which catalyzes the prenylation of many cellular signaling proteins including Ras, has been an active area of research. Much is known regarding substrate binding and the impact of various catalytic site residues on catalysis. However, the molecular level details regarding the conformational rearrangement of farnesyldiphosphate (FPP), which has been proposed via structural analysis and mutagenesis studies to occur prior to the chemical step, is still poorly understood. Following on our previous computational characterization of the resting state of the FTase ternary complex, the thermodynamics of the conformational rearrangement step in the absence of magnesium was investigated for the wild type FTase and the Y300Fbeta mutant complexed with the peptide CVIM. In addition, we also explored the target dependence of the conformational activation step by perturbing isoleucine into a leucine (CVLM). The calculated free energy profiles of the proposed conformational transition confirm the presence of a stable intermediate state, which was identified only when the diphosphate is monoprotonated (FPP2-). The farnesyl group in the computed intermediate state assumes a conformation similar to that of the product complex, particularly for the first two isoprene units. We found that Y300beta can readily form hydrogen bonds with either of the phosphates of FPP. Removing the hydroxyl group on Y300beta does not significantly alter the thermodynamics of the conformational transition, but shifts the location of the intermediate farther away from the nucleophile by 0.5 A, which suggests that Y300beta facilitate the reaction by stabilizing the chemical step. Our results also showed an increased transition barrier height for CVLM (1.5 kcal/mol higher than that of CVIM). Although qualitatively consistent with the findings from the recent kinetic isotope experiments by Fierke and co-workers, the magnitude is not large enough to affect the rate-limiting step.

Figure 1

The reaction catalyzed by farnesyltransferase.

Figure 2

The substrate binding site as seen in the crystal structure 1QBQ. Zinc, HFP, CVIM and HFP binding residues are shown as licorice. α (blue) and β (red) monomers are represented as ribbon diagram. The 7.2Å gap between the attacking nucleophile and the farnesyl acceptor is highlighted with a red line.

Figure 3

The free energy profiles of the conformational activation step in FTase ternary complexes computed as a function of the distance between the center of mass of atoms C1, C2, and O1 of FPP³⁻ or FPP²⁻ and S_γ of Cys² of the target peptides. The full PMF curve of FTase/FPP³⁻/CVIM is truncated at 3.0kcal/mol to fit into plot of the other PMFs.

Figure 4

The average conformation of the farnesyl group at the intermediate state (cyan) and that found in 1KZP (grey) are shown in licorice with the backbone conformations (ribbons) of FTase superimposed. The monoprotonated diphosphate and the farnesylated peptide are omitted for clarity.

Figure 5

The average transition state conformation of CVLM (shown in CPK), FPP, and close residues (shown in Licorice).