conformational analysis of a farnesyltransferase peptide inhibitor, cvim
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Journal of Computer-Aided Molecular Design, 14: 369382, 2000.KLUWER/ESCOM 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Conformational analysis of a farnesyltransferase peptide inhibitor, CVIM
Louis CarlacciDepartment of Chemistry, CHE 305, University of South Florida, Tampa, FL 33620, U.S.A.; E-mail:firstname.lastname@example.orgReceived 3 June 1999; Accepted 22 November 1999
Key words: cluster analysis, conformational library, conformational search, conformers, ionization, MetropolisMonte Carlo, Monte Carlo simulated annealing, Ras, solvation, states
The conformational states of the peptide Cys-Val-Ile-Met (or CVIM) were computed and characterized. CVIMinhibits farnesylation of the Ras oncogene product, p21ras, at the cysteine residue of the C-terminal segment.CVIM is active in an extended conformation. A similar peptide (KTKCVFM) appears to bind the enzyme inthe Type I bend conformation. In the present study, the conformations of CVIM were computed in an aqueousenvironment with the peptide in the zwitterionic state. Solvation free energy based on solvent accessible surfacearea and a distance dependent dielectric were used in the calculations. Final conformations of multiple independentMonte Carlo simulated annealing (MCSA) conformational searches were used as starting points for MetropolisMonte Carlo (MMC) runs. Conformations saved at intervals during MMC runs were analyzed. Conformers wereseparated by interactive clustering in dihedral angle coordinates. The four lowest energy conformers correspondingto a Type I bend, extended, AB-bend, and BA-bend were within 0.3 kcal/mol of each other, and dominant in termsof population. The Type I bend and extended conformers were supported by the binding studies. The extendedconformer was the most populated. In the AB-bend conformer, A indicates the a-helix conformation of Val, andB indicates the b-strand conformation of Ile. The AB- and BA-bend conformations differed from the extendedconformation in the value of Val % and Ile %, respectively, and from the Type I bend conformation in the value ofIle % and Val %, respectively. The four lowest energy conformers were characterized in terms of energy, density oflow energy conformations (or entropy), structure, side chain rotamer fraction population, and interatomic distances.
In mammals, Ras is a 21 kDa guanine nucleotide bind-ing protein that regulates the transduction of biologicalinformation from the plasma membrane to the nucleus. Oncogenic mutations give rise to a protein thatcan bind but not hydrolyze GTP, which results in un-controlled growth . Ras association with the plasmamembrane via a covalently linked lipid group is anecessary step in the signaling process . Ras is post-translationally modified by the covalent attachment ofa farnesyl group to the sulfur atom of the Cys residueof the C-terminal sequence CAAX (A= aliphatic, andX= methionine or serine) . The enzyme called far-nesyltransferase (or FTase) catalyzes this reaction .Next, the AAX peptide is removed and the C-terminal
carboxyl is methylated . FTase is a metalloenzymecomposed of an a subunit of 49 kDa and a b subunitof 46 kDa . Inhibition of FTase provides a target fordrug design since Ras is not able to associate with theplasma membrane without the attached lipid.
The Cys-Val-Ile-Met peptide (called CVIM) is theC-terminal segment of p21ras. CVIM inhibits FTasein vitro by acting as an alternative substrate, but hasno effect on whole cells . Experimental studiesof peptide mimetics and pseudopeptides that inhibitFTase activity have been reported (see Reference 9for a list). Qian et al. demonstrated that the centraldipeptide of the CA1A2X box is essential for farne-sylation [10,11]. In the most potent inhibitor, A1A2was substituted with an aromatic spacer that does notallow a turn conformation, and the distance between
atom Cys Sg and the C-terminal carboxyl group in thepseudopeptide was close to the corresponding distancein the extended conformation of the peptide, about10.5 . The hydrophobic property of A1A2 supports acomplementary hydrophobic region in FTase. Leonardet al.  showed that a pentapeptide derivative thatlacks cysteine inhibits FTase. In an NMR study on thebinding of the KTKCVFM peptide to FTase, Stradleyet al.  observed a Type I b-turn conformation ofthe CVFM segment. The results in References 11 and13 suggest more than one binding conformation of thepeptide.
The methodology used in the conformationalanalysis on Met5-Enkephalin (Met-Enk), a five-residue peptide with sequence YGGFM , was usedwith some modifications in the present study. Thefinal conformations of multiple independent MonteCarlo simulated annealing (MCSA) conformationalsearches were used as starting points for room tem-perature Metropolis Monte Carlo (MMC) trajectoriesthat probe the potential energy surface. The peptidewas computed in an aqueous environment given by theionized peptide, a continuum solvation model, and adistance dependent dielectric of 2r. The low energyconformational states (or conformers) were severalbend conformers and a significantly populated ex-tended conformer. A low energy bend conformationsatisfied the important pharmacophoric requirementsof the morphine model of m-receptor binding. Boththe Met-Enk study and the present study analyzedconformations of the isolated peptide in various envi-ronments. Specific interactions between the peptidesand their receptors were not considered.
Clustering algorithms were used to separate con-formers and to analyze long molecular dynamics tra-jectories. The clustering algorithm that was developedand implemented in the Met-Enk study  was sim-ilar to the algorithm that was used by McKelvey et al., which followed earlier ideas of Zimmerman et al.. Peptide conformers were identified by assigningeach residue to a region of the map of #/% dihedralangles. (Dihedral angles were defined in accordancewith the IUPAC-IUB Commission on BiochemicalNomenclature .) A single #/%-map was used forall residues and for all conformers. In these studies,a clean separation of conformational states was notpossible when a cluster of conformations lies in twodifferent main chain rotamer regions (or #/% pair or& mc-rotamer regions), or when two conformationalstates lie in the same mc-rotamer region. Karpen et al. analyzed conformations saved during long mole-
cular dynamics trajectories by statistical clustering inn-dimensional dihedral angle space, which produceda set of dihedral angles for each cluster, followed bymultidimensional scaling to project the clusters ontoa two-dimensional plane. Clustering in dihedral anglespace has the advantage over Cartesian space in thatthe clusters can be visualized in two-dimensional mainchain and side chain dihedral angle scatter plots. Inthis study, each conformer was interactively separatedby specifying the #/% pair, and &, rotamer regions thatselect the clusters of dihedral angle conformations.The $ rotamer regions were not interactively specified.
A conformer is defined as a group of similarconformations as determined by the values of theirdihedral angles. An mc-conformer is a group of con-formations with similar backbone dihedral angles. Amain chain/side chain (or ms-) conformer is a group ofconformations with similar backbone and side chaindihedral angles. The nomenclature for b-turns in thestudy by Richardson  is used to name the mc-conformers. In this study, the word bend followingthe turn type does not imply H-bonds between residuesimmediately adjacent to the turn residues.
The main purpose of the present study is to com-pute and characterize the lowest energy conformationsof CVIM in an aqueous environment. The computedconformations give us a better understanding of thebinding with FTase. A relationship between the im-portant conformations is proposed. The informationobtained here should be useful in the design of experi-ments to determine pharmacophoric properties. Basedon the limited number of computed low energy con-formational states and the size of CVIM, the peptideis a good test case for studies of physical properties(such as solvation and electrostatics). The knowledgeand expertise obtained in this study should be usefulin the conformational analysis of other peptides.
Coordinates and energy functionIn both the MCSA conformational search and MMCsimulations, the peptide geometry was generated bythe ECEPP/2 protocol , in which bond lengthsand bond angles are held fixed and dihedral anglesmay vary. L-Amino acids were employed. The peptideconformations were computed in an aqueous environ-ment, i.e. the peptide with ionized N- and C-termini,solvation based on solvent accessible surface area and
a distance dependent dielectric of 4r. Explicit watermolecules were not considered. Preliminary calcu-lations explored the use of various values for thedielectric constant.
Total energy, F, is the sum of the peptide confor-mational energy, E, and the solvation free energy, J,and is given by
F = E + J (1)Conformational energy is the sum of Lennard-Jonesnon-bonded, electrostatic and torsion potentials .CHARMm parm20 all atom energy parameters were employed. Hydrogen bond energy is built intothe electrostatic charges centered on the nuclei.
Solvation free energy is given by
whereAi is the solvent accessible surface area of atomi and si is the atom solvation parameter. Solvation pa-rameters used were the SRFOPT parameter set and the parameters for charged atoms reported in Ref-erence 23. Solvent accessible surface areas of heavyatoms were computed using MSEED  with a 1.4 probe radius.
The ECEPP geometric parameters should be trans-ferable between parameter sets since th