Aromatic Aromatic Interactions in Crystal Structures o - PDF document

AromaticInteractionsinCrystalStructuresofHelicalPeptideScaffoldsContainingProjectingPhenylalanineSubrayashastryAravinda,NarayanaswamyShamala,*ChittaranjanDas,ArumugamSriranjini,IsabellaL.Karle,*andPadmanabhanBalaram*ContributionfromtheDepartmentofPhysics,MolecularBiophysicsUnit,andDepartmentofInorganicandPhysicalChemistry,IndianInstituteofScience,Bangalore-560012,India,andLaboratoryfortheStructureofMatter,alResearchLaboratory,Washington,DC20375-5341ReceivedJanuary11,2003;E-mail:pb@mbu.iisc.ernet.in;shamala@physics.iisc.ernet.in aromaticinteractionsbetweenphenylalaninesidechainsinpeptideshavebeenprobedbythestructuredeterminationincrystalsofthreepeptides:Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe,;Boc-Aib-Ala-Phe-Aib-Phe-Ala-Val-Aib-OMe,.X-raydiffractionstudiesrevealthatallthreepeptidesadopthelicalconformationsinthesolidstatewiththePhesidechainsprojectingoutward.InterhelixassociationinthecrystalsispromotedbyPheinteractions.Atotalof15uniquearomaticpairshavebeencharacterizedinthethreeindependentcrystalstructures.Inpeptides,thearomaticsidechainslieonthesamefaceofthehelixat4positionsresultinginbothintrahelixandinterhelixaromaticinteractions.Inpeptide,thePhesidechainsareplacedontheoppositefacesofthehelix,resultinginexclusiveintermoleculararomaticinteractions.Thedistancesbetweenthecentroidsofaromaticpairrangesfrom5.11to6.86…,whilethedistanceofclosestapproachofringcarbonatomsrangesfrom3.27to4.59….ExamplesofT-shapedandparallel-displacedarrangementsofaromaticpairsareobserved,inadditiontoseveralexamplesofinclinedarrangements.Theresultssupporttheviewthattheinteractionpotentialforapairofaromaticringsisrelativelybroadandruggedwithseveralminimaofsimilarenergies,separatedbysmallactivationbarriers. aromaticinteractionsweresuggestedtobeastabilizingforceindeterminingglobularproteinstructures,fromananalysisofthefrequencyofoccurrenceofaromaticpairsinproteininteriors.BurleyandPetskoobservedªthatonanaverageabout60%ofaromaticsidechainsinproteinsareinvolvedinaromaticpairs,80%ofwhichformnetworksofthreeormoreinteractingaromaticsidechains.Phenylringcentroidsareseparatedbyapreferentialdistanceofbetween4.5and7…,anddihedralanglesapproaching90aremostAlargenumberofsubsequentanalyseshavereemphasizedtheimportanceofaromaticinteractionsinstruc-turestabilizationandhavepointedtotheoccurrenceofseveralalternatearrangementsofcloselypackedflataromaticrings.ThemostcommonidealizedarrangementsareschematicallyillustratedinFigure1,andtheparametersusedtodescriberingorientationsarealsodefined.Earlysurveysofaromaticpairsinproteinsrevealedapreponderanceofperpendicularedgetofaceorientations(Figure1d,e),althoughitwasnotedthatinwell-packedinteriorsªinteractionwithothersidechainscaninterferewithandobviouslyovercomethepreferenceforaperpendicularinteractioninaromaticpairsº.Alargebodyofexperimentalandtheoreticalworkonbenzenedimersboththeparallel-displaced(Figure1b)andT-shapedclusters DepartmentofPhysics,IndianInstituteofScience.MolecularBiophysicsUnit,IndianInstituteofScience.DepartmentofInorganicandPhysicalChemistry,IndianInstituteofNavalResearchLaboratory.(1)Burley,S.K.;Petsko,G.A.,23(2)Singh,J.;Thornton,J.M.FEBSLett(3)Burley,S.K.;Petsko,G.A.J.Am.Chem.Soc.,7995(4)Burley,S.K.;Petsko,G.A..ProteinChem,125(5)Hunter,C.A.;Singh,J.;Thornton,J.M.J.Mol.Biol.,837(6)Serrano,L.;Bycroft,M.;Fersht,A.R.J.Mol.Biol.,465 (7)McGaughey,G.B.;Gagnes,M.;Rappe,A.K.J.Biol.Chem.(8)Cox,E.G.;Cruickshank,D.W.J.;Smith,J.A.S.Proc.R.Soc.London,Ser.A21.(b)William,D.E.ActaCrystallogr74.(c)Hall,D.;Williams,D.E.ActaCrystallogr,56(9)Forexamples:(a)Janda,K.C.;Hemminger,J.C.;Winn,J.S.;Novick,S.E.;Harris,S.J.;Klemperer,W.J.Chem.Phys.,14191421.(b)Steed,J.M.;Dixon,T.A.;Klemperer,W.J.Chem.Phys,49404946.(c)Henson,B.F.;Hartland,G.V.;Venturo,V.A.;Felker,P.M.Chem.Phys,2189.(d)Ferguson,S.B.;Sanford,E.M.;Seward,E.M.;Diederich,F.J.Am.Chem.Soc,54105419.(e)Paliwal,S.;Geib,S.;Wilcox,C.S.J.Am.Chem.Soc,4497(10)Hobza,P.;Selzle,H.L.;SchlagE.W.J.Am.Chem.Soc.,3500(11)Laatikainen,R.;Ratilainen,J.;Sebastian,R.;Santa,H.J.Am.Chem.Soc,11006(12)Tsuzuki,S.;Uchimaru,T.;Mikami,M.;Tanabe,K.Chem.Phys.Lett.(13)Jaffe,R.L.;Smith,G.D.J.Chem.Phys.,2780(14)Chipot,C.;Jaffe,R.;Maiggret,B.;Pearlman,D.A.;Kollman,P.A.Am.Chem.Soc.,11217(15)Hobza,P.;Selzle,H.L.;Schlag,E.W.J.Phys.Chem.,18790(16)Sun,S.;E.R.Bernstein,E.R.J.Phys.Chem.,13348 PublishedonWeb04/15/2003J.AM.CHEM.SOC.2003,530810.1021/ja0341283CCC:$25.002003AmericanChemicalSociety (Figure1d).Indeed,whilesummarizingtheoreticalstudiesonbenzenedimers,SunandBernsteinnotethat,ªiftheoryistellingusanything,itsurelyissayingthattheinteractionpotentialbetweentwobenzenemoleculesisquiteflatandthatmanylocalminimacanexistforthedimer.Onlysmallbarriersseparateparalleldisplaced,herringbone,andperpendicular(T)conforma-Amorerecentsurveyofproteinstructuresusingalargedatasetof500proteinsconcludesthatstackinginterac-tionsareªaliveandwellinproteinsº,aconclusionbasedonananalysisofisolatedpairs(dimersofaromaticpairs).ofmeanforcecalculationleadtotheconclusionthatinanaqueousenvironmentstackingisfavored,whereasinhydro-phobicsurroundingsT-shapedstructuresaremorestable.Severalrecentexperimentalstudieshaveemphasizedtheimportanceofaromaticinteractionsinstabilizinghelicesand-hairpinsindesignedsyntheticpeptides.Thestructuralpropertiesofinteractingpairsofaromaticresidueshavealsobeeninvestigatedcomputationally.Wehavebeeninvestigatingtheeffectofaromaticresiduepositioningonhelicaland-hairpinpeptidebackbones.Theabilitytodesignconforma-tionallyrigidpeptidesofwell-definedsecondarystructureusingstereochemicallyconstrainedaminoacidshasbeenexploitedtoconstructhelicalpeptides,withappropriatelypositionedphenylalanine(Phe)sidechains.Inthisreport,wedescribethecrystalstructuresofthreehelicalpeptidescontainingmultiplearomaticrings,whichprovideanopportunitytocharacterizebothintramolecularandintermolecularPhePheinteractionsinthesolidstate.Conformationalrigidityresultingintheformationofhelicalstructureisimposedbyappropriateplace-mentofthe-aminoisobutyryl(Aib)residue.Thecrystalstructuresofthefollowingpeptidesaredescribed:containtherepeatingtetrapeptideunit(Val-3),inwhichthePheresiduesappearatpositions4andareexpectedtobealignedonthesamefaceofahelicalstructure.PeptidecontainsacentralPhe-Aib-Phe-Ala-Valsegment,whichcorrespondstoaninterchangeoftheValandPheresiduesinthecorrespondingsegmentinpeptide,resultinginanorientationofPheresidues,whichprecludesanintramolecularinteractioninhelicalstructures.StructuredeterminationinsinglecrystalsrevealshelicalconformationsforallthreepeptidesandprovidesexamplesofcooperativePhePheinteractionsthrough-outthecrystals.Perpendicular,parallel-displaced,andinclinedorientationsoftheinteractingaromaticringpairareobserved.revealsaninterestingexampleofstaticdisorderarisingfromspecificPhePheinteractionbetweenhelicalExperimentalSectionweresynthesizedbyconventionalsolutionphaseproceduresusingafragmentcondensationstrategy.BocandmethylestergroupswereusedasN-andC-terminalprotectinggroups,respectively.Peptidescouplingsweremediatedbylcarbodiimide(DCC)and1-hydroxybenzotriazole.Thepeptideswerepurifiedbyreverse-phasemedium-pressureliquidchromatography(Cm)usingmethanol/watergradients.Thepeptideswerecharacterizedby500MHzHNMRspectroscopyandMALDImass (17)Sinnokrot,M.O.;Valeev,E.;Sherrill,C.D.J.Am.Chem.Soc(18)Forexamples:(a)Bhattacharyya,R.;Samanta,U.;Chakrabarti,P.,91100.(b)Thomas,A.;Meurisse,R.;Brasseur,R.Proteins:Struct.,Funct.,Genet.,635644.(c)Thomas,A.;Meurisse,R.;Charloteaux,B.;Brasseur,R.Proteins:Struct.,Funct.,Genet634.(d)ToÂth,G.;Watts,C.R.;Murphy,R.F.;Lovas,S.Proteins:Struct.,Funct.,Genet,373381.(e)Mitchell,J.B.O.;Laskowski,R.A.;Thornton,J.M.Proteins:Struct.,Funct.Genet.,370(19)Chelli,R.;Gervasio,F.L.;Procacci,P.;Schettino,V.J.Am.Chem.Soc.,6133 (20)Butterfield,S.M.;Patel,P.R.;Waters,M.L.J.Am.Chem.Soc,9751(21)(a)Tatko,C.D.;Waters,M.L.J.Am.Chem.Soc,9372(b)Walters,M.L.Curr.OpinChem.Biol.,736(22)Schindelin,H.;Jiang,W.;Inouye,M.;Heinemann,U.Proc.Natl.Acad.Sci.U.S.A,5119(23)(a)Das,C.;Shankaramma,S.C.;Balaram.P.Eur.J.847.(b)Aravinda,S.;Shamala,N.;Rajkishore,R.;Gopi,H.N.;Balaram,P.Angew.Chem.,Int.Ed,3863(24)Gervasio,F.L.;Chelli,R.;Procacci,P.;Schettino,V.Proteins:Struct.,Funct.,Genet.,117(25)Venkatraman,J.;Shankaramma,S.C.;Balaram,P.Chem.Re(26)Kaul,R.;Balaram,P.Bioorg.Med.Chem.,105(27)Prasad,B.V.V.;Balaram,P.CRCCrit.Re.Biochem.,307(28)Karle,I.L.;Balaram,P.,6747(29)Toniolo,C.;Benedetti,E.TrendsBiochem.Sci,350(30)Balaram,P.Curr.Opin.Struct.Biol,845 Figure1.Geometriesofaromaticinteractions.Thearomaticpairsareplacedatacentroidcentroiddistanceof5.5…,whichisthedistanceatwhichthedistributionofPhePhepairsinproteinsismaximum.Fortheparallelsandwicharrangementtheenergeticallyoptimumdistanceisexpectedtobeat3.5….ThevanderWaalssurfacesgeneratedwithattachedhydrogenatomsareindicated:(a)parallelsandwich,eclipsed;(b)parallelsandwich,staggered;(c)paralleldisplaced;(d)T-shaped,edgetoface;(e)L-shaped,edgetoedge;(f)inclined.Parametersusedtodefinethearomaticinteractionareshownin(g):(…),whichisthecentroid(deg),whichistheinterplanarangle;(…),whichistheshortestdistancebetweentwocarbonatomsoftheinteractingrings. AromaticInteractionsinPeptideScaffolds J.AM.CHEM.SOC.VOL.125,NO.18,2003 spectrometry(937.1forpeptide1337.6forpeptide923.1forpeptideCrystalsofpeptideweregrownfrommethanol/watermixturesbyslowevaporation.Three-dimensionalintensitydatawerecollectedupto140usingCuKradiation(1.5418…)onaCAD4diffractometer.Thestructurewasdeterminedandrefined(SHELXS-97andSHELXL-97)toanRfactorof5.88%.Crystalsofpeptidegrownfromdioxane/methanolwereverythinandfragile.Robustprismswereobtainedbyslowevaporationfromacetonitrile/watersolution.Eventhoughmostofthecrystalscontainedcracks,theopticalextinctionwasgoodandtheX-raypeaksgenerallyhadagoodprofile.X-raydatawerecollectedforatriclinicsettingwithCuKradiation(1.5418…).Thestructurewassolvedinitiallyinspacegroup1forthetwomoleculesintheunitcell,usingavectorsearchprocedureandtangentformulaexpansionbasedona34atommodelfromthe-helicalstructureofAc-Val-Ala-Leu-Dpg-Val-Thetwomoleculesinthecellwereverysimilar,includingthedisorderinthethreephenylalanineresidues.Furthermore,theywererelatedbyarotationaxis.TheX-raydatawererecollectedinthemoresymmetricmonoclinicspacegroup2,inwhichthereisonemolecule/asymmetricunit.Inbothspacegroups,atthePhe(3),Phe(5),andPhe(11)residues,therearetwopositionsforeachphenylring,withabout50%occupancyineachposition.Becauseoftheproximityofmanyatomsofthephenylringsintheªaverageºelectrondensitymap,thesixphenylringswererestrainedtohaveidealizedvaluesforC1.39…andC2.41…andrefinedisotropically.Additionally,therearetwopositionsforthegroupintheterminal-butoxygrouprelatedbyarotationaboutaObond.Full-matrixleast-squaresrefinementresultedinanRfactorof10.2%.Crystalsofpeptideweregrownbyslowevaporationofacetonitrile/watermixtureinthetriclinicspacegroup1,withtwomoleculesintheasymmetricunit,alongwithfourwatermolecules.TheX-raydatawerecollectedonaBrukerAXSSMARTAPEXCCDdiffractometer,usingMoKradiation(0.71073…).ThestructurewassolvedbydirectmethodsandrefinedtoanRfactorof7.68%.Thecrystalanddiffractionparametersforpeptidesaresum-marizedinTable1.ResultsandDiscussionFigure2showsaviewofthemolecularconformationofthethreepeptidesdeterminedincrystals.ThebackboneandsidechaintorsionanglesaresummarizedinTable2,andhydrogenbondparametersarelistedinTable3.Peptidespredominantlyapatternofa-helical(51hydrogenbonds)withgood41hydrogenbonds(3-helicalturns)observedonlyatthetermini.TheobservedhydrogenbondslistedinTable3arebasedonacomparisonofalltheparametersforboth4and51interactions.Thestructureofanªaveragedºmoleculeofpeptide2-folddisorderisshowninFigure2b.Allthevalinesidechainsareononesideofthehelix,andallthedisorderedphenylalanine (31)(a)Sheldrick,G.M.SHELXS97,ProgramforAutomaticSolutionofCrystal;UniversityofGoÈttingen:GoÈttingen,Germany,1997.(b)Sheldrick,G.M.SHELXL97,ProgramforcrystalstructurerefinementUniversityofGoÈttingen:GoÈttingen,Germany,1997.(32)Nordman,C.E.;Nakatsu,K.J.Am.Chem.Soc,353(33)Egert,E.;Sheldrick,G.M.ActaCrystallogr,262(34)Karle,J.ActaCrystallogr,182(35)Vijayalakshmi,S.;Rao,R.B.;Karle,I.L.;Balaram,P.,84 (36)Sheldrick,G.M.;Schneider,T.R.DirectMethodsforMacromolecules.MethodsinMacromolecularCrystallography;Turk,D.,Johnson,L.,Eds.;IOSPress:Amsterdam,2001;pp72(37)Datta,S.;Shamala,N.;Banerjee,A.;Balaram,P.J.Pept.Res.(38)Baker,E.N.;Hubbard,R.E.Prog.Biophys.Mol.Biol.,97 Table1.CrystalandDiffractionParametersforPeptides empiricalformulaCcrysthabitclearandrectangularprismwithcracksclearandrectangularcrystsize(mm)0.450.20.530.070.4crystallizingsolventmethanol/water/acetoneacetonitrile/wateracetonitrile/waterspacegroupcellparams(…)12.4460(12)38.779(13)10.802(3)(…)16.027(2)8.839(2)16.361(5)(…)27.704(3)23.369(5)17.853(6)(deg)90.90.116.405(5)(deg)90.104.74(2)95.535(7)(deg)90.90.93.164(6))5526.4(11)7746.52795.8(15)442molecules/asymunit112cocrystallizedsolventnonenone4watermolecules937.11337.62(923.1))1.1261.1471.135(000)201628721024radiatn(,…)CuK(1.5418)CuK(1.5418)MoK(0.71073)temp(C)212021range(deg)140.3115.253.8scantypescanspeedvariableconstantindpdtreflcns5725578410887obsdreflcns4140[)]1877[)]6562[finalR(%)5.8810.227.68finalwR2(%)16.0920.9118.58goodnessoffit()1.0731.0150.733(e…)0.640.240.45(e…no.ofrestraints/params6/60514/7685/1225data-to-paramratio6.8:12.45:15.4:1Aravindaetal. 5310J.AM.CHEM.SOC.VOL.125,NO.18,2003 sidechainsareontheotherside.TheªaveragedºmoleculerepresentstwodistinctconformerswhererotationstakeplaceabouttheCbonds.InconformerA,wherethethreephenylringspointupward,thetorsionanglesN)are74,and)forringslabeled3A,7A,and11A.InconformerB,wheretheringspointdownward,thetorsionanglesare179,167,andtrans,t)forringslabeled3B,7B,and11B.ThesidechainconformationhaveagreaterpropensityforoccurrenceforPheresiduesininproteins.crystallizedwithtwomoleculesintheasymmetricunit.MoleculeAshowedapredominately3-helical(4hydrogen-bondingpattern),whilemoleculeBrevealedamixedhelix,withtheNandCterminalsegmentstabilizedby1and51hydrogenbonds,respectively.AcomparisonofOandHOdistanceslistedinTable3suggeststhattheintramolecularhydrogenbondsinpeptidearesignificantlyweakeronanaveragethanthoseobservedinpeptidesThisobservationmaybeofsomesignificanceinthelightofthesubsequentdiscussionofthearrayofaromaticinteractionsthatstabilizethemoleculesinthecrystal.Itis (39)Chakrabarti,P.;Pal,D.Prog.Biophys.Mol.Biol. Figure2.Molecularconformationincrystalsofpeptides.Allthehydrogenbondsareshownasdottedlines:(a),Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe;(b),Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe;(c),Boc-Aib-Ala-Phe-Aib-Phe-Ala-Val-Aib-OMe. Table2.TorsionAngles(deg)forPeptides IIIIIIAIIIBIIIIIIAIIIBIIIIIIAIIIB30.8176.037.5179.3180179.8177.0179.7178.2179.6171.2179.9179.73.1170.5Aib(8)51.054.049.137.2161.1177.6168.668.5165.5 SideChainTorsionAngles(deg)IIIIIIAIIIBIIIIIIAIIIB61.1,174.2Phe(3)172.678.6(178.7)170.170.9,108.283.6,100.9(58.8,117.7)78.8,96.766.8,72.6,163.770,166.265.587.2,99.986.9,73.7(167.4)75.4,55.166.1,55.378.6,100.8107.6,69.6(74.7,65.5,167.349.0(90.9)153.0,34.2(91.6, C(OMe).ThesidechaintorsionanglevaluesofdisorderedPheresiduesinpeptidearegiveninparentheses.Esd'sAromaticInteractionsinPeptideScaffolds J.AM.CHEM.SOC.VOL.125,NO.18,2003 conceivablethatoptimizationofintermolecularinteractionsmaycompensateforweakerintrachainhydrogenbonds.HelixPacking.Incrystalsofpeptideadjacenthelicalcolumnsarepackedinantiparallelfashionalongthecrystal--axis(Figure3a).Alongthe-axishelicalcolumnsarearrangedinparallelrowsresultinginapseudohexagonalgridarrangement.Asimilarpackingmodeoftheapproxi-matelycylindricalhelicalstructureisobservedincrystalsof(Figure3b).Nosolventmoleculeswereobservedincrystalsof,withverticalcolumnsofhelicesformedexclusivelybyhead-to-tailhydrogenbonds,betweenthefreeNHandCOgroupsathelixtermini.Inpeptide,theasymmetricunitconsistsofapairofapproximatelyparallelhelices.Thehelicalcolumnsarearrangedin,necessarily,parallelfashioninthetricliniccrystal(Figure4a).Asmanyasfourwatermoleculesarelocatedinthehead-to-tailregionprovidinghydrogenbonds,whichholdhelicalcolumnstogetherandalsofacilitatetheinteractionofadjacentcolumns(Figure4b).Figures57provideaschematicviewofthethreehelicalpeptidemolecules,illustratingtheinteractionbetweenthePhesidechains.Thecentroidcentroiddistancesaremarked.Intheinteractionsarebothintra-andinterhelical.InpeptideanarrayofintermolecularPhePhecontactsbridgeshelicalpeptidesinthecrystals.AsnotedearlierinthePhesidechainsadoptstwodifferentconformations(conformersAandB)asshowninFigure6a.Theyaredistinctfromeachother.Thephenylringsmustallpointinthesamedirectionforspatialconsiderations,thatis,toavoidcollisionswitheachother.ThedirectionsinwhichthePheringspointappeartobeequallyacceptable.AromaticInteractions.Figure8summarizestherelativeorientationofthe15uniquearomaticpairsstructurallycharacterizedinpeptides.Threeparametershavebeenusedtodescribethearomaticpairgeometries:(…),whichisthecentroidcentroiddistance.(deg),whichistheinterplanarangle,and(…),whichistheshortestdistancebetweentwocarbonatoms.ItisevidentfromtheFigure8thattwoexamplesinpeptide1/2)andPhe(7)(1/2)(Figure8b,c)correspondtoparallel-displacedconformationswithaverylowvalueof.Fourexamples,Phe-1/2,2)andPhe(11A)-1/2,2)inpeptide8e,k)andPhe(3)B()andPhe(5)A-)inpeptide(Figure8l,n)areclosetotheT-structureinwhichthebenzeneringsareapproximatelyperpendicularandarrangedinanedge-to-facemanner,withgreaterthan75.Oftheremainingnineexamples,twohaverelativelylargevalues()Phe(5)B()andPhe(3)A(1)inpeptide(Figure8m,o)andcouldbebroadlyclassifiedundertheTcategory.TheremainingsevenexamplesPhe(3)()inpeptide8a)andPhe(11B)(1/21/2,21/2,2),Phe(3A)(1/2,2),Phe(7A)(1/2,2),Phe-),Phe(7A)(),andPhe(11A)()inpeptide(Figure8d,fj)havevaluesbetween25and50,corre-spondingtoinclinedarrangements.Itmaybenotedthatthevalueof2.31…obtainedfor7A3Binteractioninpeptideisunacceptablyclose.ThetemperaturefactorsforC7D,C7E,andC7Z(conformerA)arehighindicatingsubstantialuncer-taintyinthecrystallographicallydeterminedpositions.Itshouldbenotedthatasmallrotation()abouttheaxisthroughthe1and4positionsofthebenzenering7Arelievestheclose3Bapproachwithoutchangingthecentroid (40)(a)Karle,I.L.ActaCrystallogr.,341356.(b)Karle,I.L.,157180.(c)Karle,I.L.Acc.Chem.Res.,693 Table3.HydrogenBondsforPeptides typedonoracceptor:IntermolecularBondsN(1)O(6)3.0882.232158.6157.5173.8N(2)O(7)2.8722.040155.3153.4162.4:IntramolecularBonds1N(3)O(0)2.9932.397114.5125.4126.81N(4)O(0)3.1772.331155.2158.4167.61N(5)O(1)2.9592.114161.9164.5167.21N(6)O(2)3.1932.383137.5142.8157.31N(7)O(3)2.9312.092161.3164.6165.21N(8)O(4)3.0582.204151.1153.2172.3:IntermolecularBondsN(1)O(10)2.9242.244142.3155.0132.0N(2)O(11)2.9052.070148.1146.9153.8:IntramolecularBonds1N(3)O(0)2.9852.367118.4129.4125.91N(4)O(0)3.1952.307155.6158.6168.91N(5)O(1)2.9072.030161.0164.1164.51N(6)O(2)3.0902.254134.1141.1154.41N(7)O(3)3.0692.178158.0158.8170.31N(8)O(4)3.0642.184151.7155.4165.61N(9)O(5)2.9632.103155.5159.7159.71N(10)O(6)3.1052.251144.7149.5158.41N(11)O(7)3.1542.397149.9159.6141.81N(12)O(9)3.3652.484102.1105.4166.3,Molecule:IntermolecularBondsO(7)2.9832.142153.1155.0166.0O4w2.9112.082161.6,Molecule:IntramolecularBonds1N(3)O(0)2.9762.172125.0129.7155.71N(4)O(1)3.0642.408118.7127.8133.51N(5)O(2)3.1652.63599.6111.8121.11N(6)O(3)2.9942.40497.4110.4126.31N(6)O(2)3.1832.451145.3154.4143.31N(7)O(4)3.3822.593106.4112.7153.11N(8)O(5)3.2952.51196.4103.5151.9solventO3wO(6)3.188solventO4wO(7)3.248solventO3wO4w2.711,Molecule:IntermolecularBondsO(7)2.757,Molecule:IntramolecularBonds1N(3)O(0)3.0262.212128.2132.4157.91N(4)O(1)3.0612.401118.6127.5134.01N(5)O(2)3.1492.622102.1114.2120.61N(6)O(3)3.0802.52896.5109.5122.81N(6)O(2)3.1192.358148.2156.6147.61N(7)O(3)3.2292.436158.1161.7153.51N(8)O(4)3.1662.642138.1149.3120.4solventN(2)O1w2.9322.099162.9solventO1wO2w3.476solventO3wO(5)2.877solventO4wO(6)2.931 SymmetricallyrelatedbyrelatedbySymmetricallyrelatedbySymmetricallyrelatedbyAravindaetal. 5312J.AM.CHEM.SOC.VOL.125,NO.18,2003 distance.Tofurtherincreasethedistancebetween7Aand3B,asimilarrotationcanalsotakeplaceinring3B.Theresultssuggestthatthereisnodominantpreferenceforaspecificgeometricalarrangementoftheproximalaromaticringsinthesestructures.However,someoftheobservedarrangementscorrespondcloselytotheT-shapedandparallel-displacedstructurescommonlyobservedinproteinsandpos-tulatedasenergyminimaintheoreticalcalculations.Thearrangementoftwoproximalaromaticringsisexpectedtobedominatedbyquadrupolequadrupoleinteractions,whichareanticipatedtohaveastrongangulardependence.interestingobservationthatquadrupolequadrupoleinteractionsmaybedestabilizinginafacetofacegeometryhasbeenThepresentobservationsreinforceaviewthat Figure3.Molecularpackingofpeptideincrystals:(a),Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe;(b),Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe.Thearrowsindicatethedirectionofthehelixaxes. Figure4.(a)Molecularpackingof,Boc-Aib-Ala-Phe-Aib-Phe-Ala-Val-Aib-OMe,incrystals.Arrowsindicatethedirectionofthehelixaxes.(b)Environmentofthewatermoleculeswhichholdhelicalcolumnstogetherandprovidestabilizingintracolumnandintercolumninteractions.AromaticInteractionsinPeptideScaffolds J.AM.CHEM.SOC.VOL.125,NO.18,2003 isemergingfromcomputationalstudiesofbenzenedimersthatthepotentialenergysurfaceforaninteractingpairofaromaticringisrelativelyflatwithlocalminimawhichareseparatedbyverysmallenergybarriers.Aparticularinterestingconsequenceofthearomaticinterac-tionsoncrystalpackingisobservedinthestructureofpeptide.ThethreearomaticringsofthePhesidechainsatpositions3,7,and11arealignedonthesamefaceofthehelix.OpposingantiparallelcolumnofhelicesinteractthroughtheprojectingaromaticsidechainsasschematicallyillustratedinFigure6b,c.Aninteractingladderofphenylalaninesidechainsispresentwithopposingpairsformingrungsoftheladder.Closeap-proachesofaromaticringsonthesamemoleculecorrespondingtoastabilizinginteractionbetweentherungsoftheladderarealsoobserved.Crystallographically,thethreePhesidechainsoccupytwodistinctspatialpositions,characterizedbydistinct (41)(a)Brown,N.M.D.;Swinton,F.L.J.Chem.Soc.,Chem.Commun770.(b)Hunter,C.A.;Sanders,J.K.M.J.Am.Chem.Soc.5534.(c)Hunter,C.A.;Lawson,K.R.;Perkins,J.;Urch,C.J.Chem.Soc.,PerkinTrans.2,651 (42)Shetty,A.S.;Zhang,J.;Moore,J.S.J.Am.Chem.Soc,1019 Figure5.(a)SchematicviewofthePhePheinteractionsincrystalsofpeptide.Themolecularbackboneisshownasaribbonrepresentation,andthecentroidtocentroiddistances(…)betweenproximalaromaticringsaremarked.(b)SchematicviewofaproximalPhePheinteractiononthesurfaceofthehelixinpeptide.TheresiduesshownarePhe(3)andPhe(7).TherepresentationwasgeneratedusingtheprogramMOLMOL. Figure6.SchematicviewofthePhePheinteractionsincrystalsofpeptide:(a)twosetsofPheconformationsseparatelyindicatedonahelicalscaffold;(b,c)twoalternatesetsofPhePheinteractionsobservedbetweenhelicalmoleculeswhichformantiparallelcolumnsinthecrystals.Thecentroidtocentroiddistances(…)areindicated.Aravindaetal. 5314J.AM.CHEM.SOC.VOL.125,NO.18,2003 Phesidechainconformations.ParticularlynoteworthyisthefactthatinopposingcolumnsofhelicesifthePhesidechainatpositions3,7,and11adoptsconformationA,thenthefacingsetofPheresiduesadoptsconformationB,aclearindicationthatspecificarrangementsofaromaticringsstabilizeinterhelixpacking.Thereis,however,nolong-rangecorrelationbetweentheringconformationsinthecrystalresultinginastatisticaldistributionofhelicalcolumnpairs,suchthatonanaveragehalfthemoleculesadoptconformationA,whiletheotherhalfadoptsconformationB,resultinginanoccupancyfactorof0.5fortheindividualconformations.Interestingly,theisopropylsidechainsofValresidues,whichoccurontheotherfaceofthehelicalmolecules,arenotdisorderedinthecrystals.Thisstudyillustratestheuseofhelicalpeptidescaffoldsforprobingthenatureofaromaticsidechaininteractionsindesignedpeptides,wherethespatialdispositionofthesidechainscanbecontrolledbyplacingaromaticresiduesatappropriatesequencepositions.ThecrystallinityofhydrophobichelicalpeptidescontainingAibresidues,whichactasstereochemicaldirectorsofpeptidechainfolding,permitsprecisestructuralcharacterizationoftheseinteractionsbyX-raydiffraction.Theanalysisof15uniquearomaticpairsinthreeindependenthelicalpeptidecrystalstructuresprovidesexamplesofT-shaped,parallel-displaced,andinclinedarrangementofinteractingPherings.Theexperimentalobservationsaregenerallyconsistentwiththeviewthattheenergylandscapeforapairofinteractingphenylringsconsistsofabroad,relativelyflatminimum,whichappearstobesomewhatrugged,withseverallocalminimaseparatedbysmallenergybarriers.Incrystals,cooperativeinteractionsbetweenaromaticringscanleadtointerestingexamplesofdisorderinthesolidstate,asexemplifiedbythestructureofthedodecapeptidecontainingthreePheresidues.Cooperativearomaticinteractionsmayprovetobeanimportantdeterminantofbiologicalstructuresandhavebeenrecentlysuggestedtobeimportantinself-assemblyofamyloidfibrils.AninterestingrecentcrystalstructureofthetetrapeptidePhe-Gly-Phe-Glyrevealsafullyextended,flat,sheetconformation.Theabsenceofbucklingorpleatingofthepeptidechainmayhaveitsoriginintheobservednetworkofaromaticinteractioninthecrystals.ThisworkwassupportedbyaprogramgrantintheareaofDrugandMolecularDesignbytheDepartmentofBiotechnology,agrantfromtheCouncilofScientificandIndustrialResearchofIndia.TheCCDdiffrac-tometerfacilitywassupportedbytheIRPHAprogramoftheDepartmentofScienceandTechnologyofIndia.TheworkatNavalResearchLaboratorywassupportedbytheNationalInstitutesofHealth,GrantGM-30902,andtheOfficeofNavalSupportingInformationAvailable:X-raycrystallographicfilesforpeptide(CIFformat).ThismaterialisavailablefreeofchargeviatheInternetathttp://pubs.acs.org. (43)(a)Gazit,E.FASEBJ,7783.(b)Gazit,E.Curr.Med.Chem,1667(44)Birkedal,H.;Schwarzenbach,D.;Pattison,P.Chem.Commun,2812(45)IUPACIUBCommissiononBiochemicalNomenclature.,3471(46)Koradi,R.;Billeter,M.;Wuthrich,K.J.Mol.Graphics,51 Figure7.SchematicviewofthePhePheinteractionsobservedincrystalsofpeptide.Thecentroidtocentroiddistances(…)areindicated.NotethattherearenointramolecularPhePheinteractionsinthispeptide. Figure8.Summaryoftheuniquearomaticaromaticinteractionsobservedinthecrystalsofpeptides.Theparameters,andindicated:(ac)peptide;(dk)peptide;(lo)peptide.TheinteractingPhesidechainsareshownasbenzylgroupswithattachedhydrogens.ThevanderWaalssurfacesareshown.AromaticInteractionsinPeptideScaffolds J.AM.CHEM.SOC.VOL.125,NO.18,2003