ABSTRACT: Studies aimed at elucidating the unknown Mg2+ binding site in protein farnesyltransferase (FTase) are reported. FTase catalyzes the transfer of a farnesyl group to a conserved cysteine residue (Cys1p) on a target protein, an important step for proteins in the signal transduction pathways (e.g., Ras). Mg2+ ions accelerate the protein farnesylation reaction by up to 700-fold. The exact function of Mg2+ in catalysis and the structural characteristics of its binding remain unresolved to date. Molecular dynamics (MD) simulations addressing the role of magnesium ions in FTase are presented, and relevant octahedral binding motifs for Mg2+ in wild-type (WT) FTase and the D?352A mutant are explored. Our simulations suggest that the addition of Mg2+ ions causes a conformational change to occur in the FTase active site, breaking interactions known to keep FPP in its inactive conformation. Two relevant Mg2+ ion binding motifs were determined in WT FTase. In the first binding motif, WT1, the Mg2+ ion is coordinated to D352?, zinc-bound D297?, two water molecules, and one oxygen atom from the ?- and ?-phosphates of farnesyl diphosphate (FPP). The second binding motif, WT2, is identical with the exception of the zinc-bound D297? being replaced by a water molecule in the Mg2+ coordination complex. In the D?352A mutant Mg2+ binding motif, D297?, three water molecules, and one oxygen atom from the ?- and ?-phosphates of FPP complete the octahedral coordination sphere of Mg2+. Simulations of WT FTase, in which Mg2+ was replaced by water in the active site, recreated the salt bridges and hydrogen-bonding patterns around FPP, validating these simulations. In all Mg2+ binding motifs, a key hydrogen bond was identified between a magnesium-bound water and Cys1p, bridging the two metallic binding sites and, thereby, reducing the equilibrium distance between the reacting atoms of FPP Cys1p. The free energy profiles calculated for these systems provide a qualitative understanding of experimental results. They demonstrate that the two reactive atoms approach each other more readily in the presence of Mg2+ in WT FTase and mutant. The flexible WT2 model was found to possess the lowest barrier toward the conformational change, suggesting it is the preferred Mg2+ binding motif in WT FTase. In the mutant, the absence of D352? makes the transition toward a conformational change harder. Our calculations find support for the proposal that D352? performs a critical role in Mg2+ binding and Mg2+ plays an important role in the conformational transition step.
