Conformational analysis of a farnesyltransferase peptide inhibitor, CVIM

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<ul><li><p>Journal of Computer-Aided Molecular Design, 14: 369382, 2000.KLUWER/ESCOM 2000 Kluwer Academic Publishers. Printed in the Netherlands.</p><p>369</p><p>Conformational analysis of a farnesyltransferase peptide inhibitor, CVIM</p><p>Louis CarlacciDepartment of Chemistry, CHE 305, University of South Florida, Tampa, FL 33620, U.S.A.; E-mail:lou@finch.cas.usf.eduReceived 3 June 1999; Accepted 22 November 1999</p><p>Key words: cluster analysis, conformational library, conformational search, conformers, ionization, MetropolisMonte Carlo, Monte Carlo simulated annealing, Ras, solvation, states</p><p>Summary</p><p>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.</p><p>Introduction</p><p>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[1]. Oncogenic mutations give rise to a protein thatcan bind but not hydrolyze GTP, which results in un-controlled growth [2]. Ras association with the plasmamembrane via a covalently linked lipid group is anecessary step in the signaling process [3]. 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) [4]. The enzyme called far-nesyltransferase (or FTase) catalyzes this reaction [5].Next, the AAX peptide is removed and the C-terminal</p><p>carboxyl is methylated [6]. FTase is a metalloenzymecomposed of an a subunit of 49 kDa and a b subunitof 46 kDa [7]. Inhibition of FTase provides a target fordrug design since Ras is not able to associate with theplasma membrane without the attached lipid.</p><p>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 [8]. 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</p></li><li><p>370</p><p>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. [12] showed that a pentapeptide derivative thatlacks cysteine inhibits FTase. In an NMR study on thebinding of the KTKCVFM peptide to FTase, Stradleyet al. [13] observed a Type I b-turn conformation ofthe CVFM segment. The results in References 11 and13 suggest more than one binding conformation of thepeptide.</p><p>The methodology used in the conformationalanalysis on Met5-Enkephalin (Met-Enk), a five-residue peptide with sequence YGGFM [14], 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.</p><p>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 [14] was sim-ilar to the algorithm that was used by McKelvey et al.[15], which followed earlier ideas of Zimmerman et al.[16]. 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 [17].) 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&amp; mc-rotamer regions), or when two conformationalstates lie in the same mc-rotamer region. Karpen et al.[18] analyzed conformations saved during long mole-</p><p>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 &amp;, rotamer regions thatselect the clusters of dihedral angle conformations.The $ rotamer regions were not interactively specified.</p><p>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 [19] 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.</p><p>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.</p><p>Methodology</p><p>Coordinates and energy functionIn both the MCSA conformational search and MMCsimulations, the peptide geometry was generated bythe ECEPP/2 protocol [20], 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</p></li><li><p>371</p><p>a distance dependent dielectric of 4r. Explicit watermolecules were not considered. Preliminary calcu-lations explored the use of various values for thedielectric constant.</p><p>Total energy, F, is the sum of the peptide confor-mational energy, E, and the solvation free energy, J,and is given by</p><p>F = E + J (1)Conformational energy is the sum of Lennard-Jonesnon-bonded, electrostatic and torsion potentials [20].CHARMm parm20 all atom energy parameters [21]were employed. Hydrogen bond energy is built intothe electrostatic charges centered on the nuclei.</p><p>Solvation free energy is given by</p><p>J =i</p><p>Aisi (2)</p><p>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 [22]and the parameters for charged atoms reported in Ref-erence 23. Solvent accessible surface areas of heavyatoms were computed using MSEED [24] with a 1.4 probe radius.</p><p>The ECEPP geometric parameters should be trans-ferable between parameter sets since they are based onthe equilibrium X-ray conformations of proteins. Thesolvation parameters should be transferable becausethey were obtained from small molecule data.</p><p>MCSA and MMC protocols</p><p>MCSA conformational searches and MMC simula-tions used the program Salp/2, which is an enhancedversion of the program that was used in Reference 14.Conformational searches consisted of multiple inde-pendent MCSA runs. The final conformation of eachMCSA run was the starting point for an MMC run.The enhanced MCSA methodology is described next,followed by a summary of program enhancements,and a description of the MMC methodology.</p><p>An MCSA calculation looped through 300 in-dependent conformational search runs, each startingfrom a random conformation. In the assignment of di-hedral angle values to the 300 random starting confor-mations, side chain $ angles were randomly assignedfrom a rotamer library backbone &amp; angles were set to180; and backbone # and % angles were randomlyassigned in the range 180 by the use of a ran-dom number generator. The nomenclature of dihedralangles is in accordance with Reference 17.</p><p>Each MCSA run consisted of equilibration fol-lowed by prediction. Separate Boltzmann tempera-ture factors, RT (in kcal/mol), were used to evaluatemain chain and side chain trial conformations, in or-der to make the transition between random and foldedstates occur at about the same place in the anneal-ing. An MCSA run started out with high Boltzmanntemperature factors. As the Boltzmann temperaturefactors are lowered during an MCSA run, the con-formational search is restricted to lower energy path-ways. MCSA protocol parameters are summarized inTable 1.</p><p>An MCSA run consisted of a specified number ofannealing steps. In an annealing step, a specified num-ber of trial conformations (NCON) were sequentiallyevaluated. NCON was specified for the first equilibra-tion step, the remaining equilibration steps, and theprediction steps.</p><p>In trial conformation generation, one of #, %, &amp;or $ was randomly selected; and a new value wasrandomly assigned in the range PERT of the cur-rent value. PERT is the perturbation. Certain dihedralangles were varied in pairs. Selection of # (or %) ofresidue i resulted in trial values for both #i and %i1(or %i and #i+1). Selection of $1 or $2 resulted in trialvalues for both.</p><p>An MCSA cycle is defined as the process of evalu-ating a trial conformation. First, a trial conformation isgenerated; and the total energy computed. The energyof the trial conformation is compared to the energyof the previously accepted conformation. Trial con-formations are accepted or rejected by the use of theMetropolis criteria [25]. The most recently acceptedtrial conformation is used in the next cycle.</p><p>Except for the first and last steps of an MCSArun, the Boltzmann temperature factors were reducedat the end of each MCSA cycle. In the first step, aconstant temperature helps remove steric interactionspresent in the random starting conformation. In thelast step, the peptide conformation is optimized atthe low temperature. Lowering Boltzmann tempera-ture factors at the end of an MCSA cycle allows slowannealing in the region where the peptide folds. Thesimulated annealing consisted of two sequential pe-riods of exponential cooling. Boltzmann temperaturefactors were specified for the first and last equilibra-tion step, RTeqi and RTeqf, and the last predictionstep, RTprf. Plots of RT versus number of trial con-formations gave plateaus as RT approached RTeqfand RTprf. The current Boltzmann temperature factorswere reduced through division by a parameter called b</p></li><li><p>372</p><p>Table 1. Protocol used in MCSA and MMC runs</p><p>Parametera MCSA MMC</p><p>Equilibrationb Predictionc Equilibrationb Productionc</p><p>NSTEP 11 14 1 1NCON 1500 1500 15 000 25 000</p><p>(3000 in step 1)RT main chain 8.0 to 4.0 4.0 to 0.6 0.6 0.6RT side chain 4.0 to 2.0 2.0 to 0.6 0.6 0.6NPRCHK 2 (5 in step 1) 2 6 10</p><p>Parametera MCSA MMC</p><p>PERT 180 backbone, 180 side chain 10 backbone, 30 side chainXACRAT 35% main chain trial conformations 40% main chain trial conformations</p><p>40% side chain trial conformations 40% side chain trial conformationsPERTRD 1.7 for both main chain and side 1.5 for both main chain and side chain</p><p>chain perturbations</p><p>aNSTEP is the number of steps. NCON is the number of trial conformations evaluated in the step. RTis the Boltzmann temperature factor in kcal/mol. NPRCHK is the number of times the acceptanceratio is checked in each step. PERT are the initial perturbations used to generate trial conformations.XACRAT is the acceptance ratio limit. PERTRD is the factor used to lower the perturbation.</p><p>bParameters for equilibration.cParameters of MCSA prediction or MMC production.</p><p>defined for both main chain and side chain RT. Thevalues of b used during equilibration and prediction,beq and bpr, respectively, are given by</p><p>beq = {RT eqi/RT eqf }(1/(NCONeq(NST EPeq 1) 1)</p><p>andbr = {RTeqf /RTprf } (1/(NCONpr</p><p>(NSTEPpr 1)) (3)The fraction of trial conformations accepted</p><p>(called the acceptance ratio) was determined after aspecified number of trial conformations were evalu-ated. If the acceptance ratio fell below the acceptanceratio limit, the perturbation was reduced by a factor of1.7. If the accep...</p></li></ul>

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