Finding a needle in the haystack: computational modeling of Mg2+ binding in the active site of protein farnesyltransferase.

Protein farnesyltransferase (FTase) catalyzes the transfer of a farnesyl group to a cysteine residue (Cys1p) on a CaaX motif (a=aliphatic, X=Met) at or near the C-terminus of a protein substrate(1, 2) (see Figure 1). Farnesylation is a critical step in the posttranslational modification of the Ras family of proteins(3–7). Mutations in these proteins are associated with almost 50% of tumors in human-colon-carcinomas and 30% of human cancers. The potential to prevent unmitigated cell growth by inhibiting mutant Ras activity has made the FTase catalyzed farnesylation reaction the subject of numerous studies(2, 8–24). Several FTase inhibitors (FTIs) have entered different clinic trial phases(2, 25–31). FTase is a zinc metalloenzyme composed of two subunits, with the active site located in a funnel shaped cavity within the β subunit. Computational, kinetic, structural and mutagenesis investigations have revealed the nature of this active site. The two reacting atoms, namely C1 of the substrate farnesyldiphosphate (FPP) and S γ of Cys1p, are 7 A (see Figure 2) apart in the crystal structures of the ternary complex (FTase-FPP-peptide substrate). The protein substrate coordinates to the zinc ion through S of Cys1p, forming a tetrahedral zinc complex along with the enzymatic residues D297β, C299β and H362β. The direct coordination of the reactive sulfur atom to the zinc ion, suggests that the zinc ion is involved in the catalytic reaction(32). The exact nature of the reaction pathway has been a source of debate. Recent kinetic isotope effects (KIE) experiments by the Fierke (33) and Distefano groups (20) have indicated that the reaction adopts a SN2-like associative pathway mechanism, with partial carbocation character (SN1-like) in the transition state structure. Figure 1 The FTase catalyzed farnesylation reaction. Figure 2 Snapshot of the FTase active site from the initial structure. Zinc is represented as a grey sphere. The black dashed line connects the two reactive atoms, C1 of FPP and S γ of the cysteine peptide (Cys1p) and is greater than 7A in length. ... Mg2+ ions have been found to enhance the rate of product formation by ~700-fold without affecting the affinity of either the peptide or FPP(34). The exact function of Mg2+ ions has been a subject of recent interest, and is not entirely understood. A magnesium ion has been determined to be bound in the active site, though the exact nature of its binding remains to be resolved by structural analysis. Kinetic and mutagenesis experiments performed by Fierke and coworkers(34), in conjunction with crystallographic studies of FTase with Mn2+ ions carried out by Beese and coworkers(2), have provided insights into the characteristics of Mg2+ ion binding in the active site. It is widely accepted that bound Mg2+ forms an octahedral complex with negatively charged groups. In a proposed model, Mg2+ coordinates to a non-bridging phosphate oxygen of α- and β-phosphate in FPP, the carboxylate side chain of D352β, a carboxylic oxygen of zinc bound D297β, and a water molecule(34). D352β has been identified to be crucial for Mg2+ ion binding in the active site, with mutations (Dβ352A and Dβ352K) diminishing the rate enhancing ability of Mg2+ ions. It has been proposed that the formation of a D297β bridged bimetal catalytic site helps reduce the 7 A distance gap between the two reacting atoms, bringing the enzyme into a pre-organized state for the chemical step. It is also thought that Mg2+ ions promote the farnesylation reaction by stabilizing the developing negative charge on the diphosphate (PPi) leaving group, and increasing the carbocation character of C1 in the chemical step. Computational studies have aided in understanding the FTase catalyzed farnesylation reaction. Cui et al. have studied the substrate binding and conformational activation of FTase in the absence of Mg2+ ions using MD simulations and free energy sampling methods(17, 18). They found that the highly charged binding pocket kept FPP3− locked in an inactive conformation, and the monoprotonated diphosphate group (FPP2−) was the only from of FPP that could activate the conformational transition via rotation of the bond between the first two isoprenoid groups. The Ramos group’s computational efforts to explain the so-called “distance paradox”(35) agreed with the earlier Merz et al. study, that conformational activation is attributed to the isoprenoid-rotation mechanism. More recently, Klein and co-workers investigated the reaction pathway using a quantum mechanical/molecular mechanical (QM/MM) approach(19). Their results suggest that the reaction goes through “an associative pathway with dissociative characteristics”, in agreement with experimental observations. This study focused on the chemical step, and did not explore the prior conformational change in the system. However, the system investigated, FTase/FPP3− without Mg2+ is a system that is not capable of the necessary conformational change to bring the two reactive centers into contact. Moreover, it is a system that has not been observed experimentally because FPP3− under these conditions is likely protonated. Crucially, in all of the studies reported to date the role of Mg2+ has not been examined leaving a large gap in our understanding of FTase activity under physiological conditions. Nonetheless, previous studies have demonstrated the efficacy of computational methods in exploring FTase activity, and have opened the way to explore the catalytic reaction in the presence of Mg2+ ions. In this paper we present a computational study aimed at locating the Mg2+ binding site in FTase. We use MD simulations and free energy calculation methods to elucidate the function of Mg2+ ions in the conformational transition step of the reaction. The role of D352β in binding a magnesium ion in the active site is also investigated in this study. We perform calculations on WT FTase and the Dβ352A single mutant with FPP3− and CVIM peptide as substrates, in the presence of an active site bound Mg2+ ion. The Dβ352A mutation was examined in order to reproduce and provide molecular-level insights into experimental results. We validate our simulations by replacing the active site bound Mg2+ ion with a water molecule, and compare the results from this simulation to previously published results. In the absence of crystal structures of FTase with bound Mg2+ ion in the active site, we generate starting structures using a methodology successfully utilized earlier by Merz(36) to identify CO2 binding sites in human carbonic anhydrase II. We start with several “educated” guesses as starting structures for WT and mutant FTase/FPP3−/Mg2+ complexes based on experimental data. We then identify relevant Mg2+ binding motifs and investigate them using MD simulations. We analyze our simulations for hydrogen-bonding patterns, and conformational changes across these motifs. We also calculate the potential of mean force (PMF) for the conformational change in FPP for every motif. The results from our investigations provide new insight into the role of Mg2+ ions in helping FPP undergo the conformational transition in the farnesylation reaction, and the associated experimentally observed rate enhancement. We identify a critical hydrogen-bonding pattern in WT FTase, and test the proposal for a D297β bridged bi-metallic catalytic site. These simulations provide a physical basis for the function of D352β in FTase, both with and without magnesium ions. Based on the results from our calculations, we propose a novel model for Mg2+ binding in the active site of FTase that can be experimentally tested. The following section of this paper introduces the computational methods used to study the FTase reaction. In this section, we describe methods used to generate and investigate possible Mg2+ binding motifs. In Section 3, we present our analyses of the various systems. In the first part of this section, we present the results from modeling mutant and WT FTase with Mg2+ ions. The second part of this section analyzes the results from our simulation without bound magnesium in the active site. In the third part of the section, we present the results from our free-energy calculations. Our conclusions from this study are presented in Section 4. We explain the impact of mutations on the enzyme and, finally, we describe our proposed motif for Mg2+ binding in the FTase active site.