Computational Studies of the Farnesyltransferase Ternary Complex Part I:  Substrate Binding

Abstract

Farnesyltransferase (FTase) catalyzes the transfer of farnesyl from farnesyl diphosphate (FPP) to cysteine residues at or near the C-terminus of protein acceptors with a CaaX motif (a, aliphatic; X, Met). Farnesylation is a critical modification to many switch proteins involved in the extracellular signal transduction pathway, which facilitates their fixation on the cell membrane where the extracellular signal is processed. The malfunction caused by mutations in these proteins often causes uncontrolled cell reproduction and leads to tumor formation. FTase inhibitors have been extensively studied as potential anticancer agents in recent years, several of which have advanced to different phases of clinical trials. However, the mechanism of this biologically important enzyme has not been firmly established. Understanding how FTase recruits the FPP substrate is the first and foremost step toward further mechanistic investigations and the design of effective FTase inhibitors. Molecular dynamic simulations were carried out on the ternary structure of FTase complexed with the FPP substrate and an acetyl-capped tetrapeptide (acetyl-CVIM), which revealed that the FPP substrate maintains an inactive conformation and the binding of the diphosphate group can be largely attributed to residues R291beta, K164alpha, K294beta, and H248beta. The FPP substrate assumes an extended conformation in the binding site with restricted rotation of the backbone dihedral angles; however, it does not have a well-defined conformation when unbound in solution. This is evident from multinanosecond MD simulations of the FPP substrate in a vacuum and solution. Our conclusion is further supported by theoretical J coupling calculations. Our results on the FPP binding are in good agreement with previous experimental kinetic studies on FTase mutants. The hypothetical conformational activation of the FPP substrate is currently under investigation.

Computational Studies of the Farnesyltransferase Ternary Complex Part II: The Conformational Activation of Farnesyldiphosphate

“…Previously, we carefully calibrated the force field parameters of the backbone rotations of the farnesyl group, with which we were able to compute the ensemble averaged J-coupling constant for one backbone dihedral angle in excellent agreement with experiment (26). Subsequently, a molecular dynamics (MD) simulation of the FTase ternary complex (FPP 3− and the peptide CVIM) was carried out in explicit solvent for over 6 nanoseconds, during which only the resting state was sampled and the diphosphate was locked in its inactive form by the positively charged binding pocket consisting of R291β, K294β, and K164α.…”

“…The general system setup and the protocol for the MD simulation of the FTase ternary complex (FPP and CVIM) have been described elsewhere (26), and were used in this study with little modification. As before, all the molecular dynamics simulations and potential of mean force calculations were carried out using the sander module in AMBER8 (28).…”

“…The results are available in the supplementary material. The Gaff generic AMBER force field (31) for organic compounds, with additional torsion corrections obtained previously (26), was employed for the bonding (bond, angle, and torsion) and van der Waals interactions.…”

Exploring the Role of the Active Site Cysteine in Human Muscle Creatine Kinase

“…All bonds with hydrogen atoms involved were constrained with SHAKE (42). The whole system then was equilibrated by using a protocol similar to that described in Cui et al (43). Equilibration was then followed by production runs of 13 ns and 14.5 ns for the neutral and charged Cys283 complexes, respectively, using 2 fs time steps during which snapshots were collected every 2.0 ps.…”

Molecular Dynamics Simulations: Difficulties, Solutions and Strategies for Treating Metalloenzymes

“…Examples include the metalloenzymes human carbonic anhydrase II [29,32,[37][38][39] , alcohol dehydrogenase [40,41], yeast cytosine deaminase [33], histone deacetylase [34], Zn-beta-lactamase [35,36,42,43], farnesyltransferase [44][45][46][47][48][49], urea amidohydrolase [50] , adenosine deaminase [51], Zinc and Copper binding Alzheimer's amyloid beta-peptide [52] , and the system Cu(II)-PHGGGWGQ octapeptide [53].…”