OMMUNICATIONASurveyofLeft-handedHelicesinProteinStructuresMarianNovotn - PDF document

OMMUNICATIONASurveyofLeft-handedHelicesinProteinStructuresMarianNovotnyandGerardJ.KleywegtDepartmentofCellandMolecularBiology,UppsalaUniversity,Box596,SE-75124Uppsala,SwedenLinnaeusCentreforBioinformatics,BiomedicalCentre,Box598,SE-75124Uppsala,SwedenAllnaturallyoccurringaminoacidswiththeexceptionofglycinecontainoneormorechiralcarbonatomsandcanthereforeoccurintwodifferentconÞgurations, Abbreviationsused:PDB,ProteinDataBank;EDS,ElectronDensityServer.E-mailaddressofthecorrespondingauthor:gerard@xray.bmc.uu.se doi:10.1016/j.jmb.2005.01.037J.Mol.Biol.,231–241 conformation.SomeadditionaldatacanbefoundonourwebsiteSometimesright-handedheliceshaveaSchell-manturnasaC-cap.Insuchamotif,thehelixisterminatedbyaresiduewithaleft-handedconfor-mation,and5Ð2or6Ð1hydrogenbondsareusuallyformed.Ofthepresentsetofleft-handedhelices,13containaleft-handedversionoftheSchellmanturn.Whenevertwoconsecutiveresidueshaveahelicalconformationofoppositehandedness(i.e.RLorLR),theyformamotifcalledanest.Analysisofthe31left-handedhelicesandtheirßankingresiduesrevealedthatsixofthemcontainbothanRLnestattheirNterminusandanLRnestattheirCterminus,whereastenofthemcontainneither.OnehelixonlycontainsanRLnest,whereas14helicescontainonlyanLRnest.Toassessiftheshortaminoacidsequencesoftheleft-handedhelicesalwaysoccurinsuchsecondarystructureelements,alloccurrencesofeachofthe31sequenceswerelocatedina95%sequence-identitysubsetofthePDBwiththePATINPROTserver.expected,noneofthesequencesfoundintheleft-handedhelicesshowedanystrongsecondarystructurepreferences.Mostwerefoundtooccurinallthreesecondarystructuretypes(helix,strandandloop),e.g.thesequenceAQGGoriginallyfoundinPnuclease(PDBcode1ak0)asaleft-handedhelixwasfoundintenotherproteinsinthePDBsubsetwhereitappearedasahelix,astrand,aloopandcombinationsthereof.Thesequenceofthelongestleft-handedhelix(HAGEGG)wasnotfoundinanyotherproteininthePDB.Theextenttowhichleft-handedhelicesare Table1.Left-handedhelicesencounteredinasubsetofthePDBPDBcodeandchainIDProteinnameCATHclassiÞcationResiduesSequenceReference1bd0AAlanineracemase2.40.37.1040Ð44ANAYG1autLActivatedproteinC2.10.25.10101Ð104DNGG31b9wAMerozoitesurfaceprotein1P.cynomolgiÐ52Ð55KNGG31g2lBCoagulationfactorX2.10.25.10258Ð261DNGD31kliLCoagulationfactorVII2.10.25.1094Ð97ENGG31n1iAMerozoitesurfaceprotein1P.knowlesiÐ57Ð60NNGG31ob1CMerozoitesurfaceprotein1P.falciparumÐ52Ð55NNGG31rfn_CoagulationfactorIX2.10.25.1091Ð94KNGR31pb5ALnrmodulefromNotchÐ28Ð32GWDGG31kdgACellobiosedehydrogenaseÐ532Ð535YENW31hzmAProteinphosphatase6Ð61Ð64IMLR1gtxA4-Aminobutyrateaminotransferase3.30.70.16070Ð73SQIS1qj5A7,8-Diaminopelargonicacid3.30.70.16050Ð53SSWW32gsaAGlutamatesemialdehydeaminotransferase3.30.70.16065Ð68GTWG32oatAOrnithineaminotransferase3.30.70.16083Ð86SSYS31bnlAEndostatin(Homosapiens)3.10.100.10135Ð138CETW1dy2AEndostatin(M.musculus)3.10.100.10207Ð210CEAW1koeSEndostatin(M.musculus)3.10.100.10266Ð269CETW1bqbAAureolysin1.10.390.10223Ð226DNGG1npcENeutralprotease1.10.390.10227Ð230DNGG8tlnEThermolysin1.10.390.10226Ð229DNGG1j9qANitratereductase(A.feacalis)2.60.40.240105Ð108ALGG31nifANitratereductase(A.cycloclates)2.60.40.240105Ð108ALGG31oe1ANitratereductase(A.xylosoxidans)2.60.40.24099Ð102ALGG31ptmA4-Hydroxythreonine-4-phosphatedehydrogenaseÐ211Ð216HAGEGG31ak0EP1nuclease1.10.575.10131Ð134AVGG1mzr_2,5-Diketo--gluconatereductaseÐ191Ð194AQGG31h21ASplit-soretcytochromeÐ77Ð81GGISD31hxxAOmpfporin2.40.160.10143Ð146NFFG31jv1AGlcnac1puridyltransferaseÐ182Ð185KYFG31kwsABeta-1,3-glucuronyltransferase33.90.550.10298Ð301AANCThefour-characterPDBcode,followedbythechainidentiÞer(whereanunderscoresigniÞesablankchainidentiÞer).Theentriesaresortedintheorderinwhichtheyarediscussedinthetext(i.e.groupedbyfunction).Entries1hzmand1pb5areNMRstructures;allotherstructuresweredeterminedbyX-raycrystallographicmethods.TheCATHclassiÞcation;adashindicatesthatthePDBentryhadnotbeenclassiÞedbyCATHyet.Thetypeoftheleft-handedhelices,asassignedmanuallybasedonhydrogen-bondingpatterns.C.Abergel,S.Jeudy,V.MonchoisandJ.M.Claverie,StructureofDkgAfromEscherichiacoliat2.13Aresolutionsolvedbymolecularreplacement(unpublishedresults). http://xray.bmc.uu.se/ Left-handedHelices formation.Alanineracemase(PDBcode1bd0)wasalreadyknowntocontainaleft-handedhelix.catalyticresidueisLys39andtheleft-handedhelixcoversresidues40to44,whereTyr43isthecovalentattachmentsiteforthecofactorpyridoxalphosphate(Figure2(a)).ThecarbonyloxygenatomofGly44formsaninterdomainhydrogenbondwithTheentirehelixispartofthePROSITEpattern(PS00395)forthealanineracemasefamily.EGF-likefamilyAleft-handedhelixwasidentiÞedinsevenproteinswithEGF-likedomains(PDBcodes1aut,1b9w,1g2l,1kli,1n1i,1ob1and1rfn).Fouroftheseareblood-clottingproteins(factorVII,factorIX,factorXandproteinC)andthreearemerozoitesurfaceproteinsfromvariousPlasmodiumspecies.Thesetwofunctionallydistinctgroupsshowweakbutdetectablesequencesimilarity,mainlyduetoconservedcysteineresiduesthatformdisulphidebridges,butalsoinashortstretchofsequencecontainingtheleft-handedhelix.Thehydrogen-bondingpatternofthesehelicesclassiÞesthemas-helicesratherthan-helices.ThesevenproteinshaveaconservedAsnresidueintheirleft-handedhelicesthatformsanimportanthydrogenbondeitherwithresidue5(proteinCandfactorVII)or7(merozoitesurfaceproteins)intheEGF-likedomain,orwithresiduesintheproteasedomain(factorIXandfactorX).Thereisalsosomeevidencethattheleft-handedhelixinfactorIXisinvolvedinthebindingoffactorVIII,whichisitsnaturalinteractionpartnerintheblood-clottingcascade.DoublemutationofAsn89andAsn92toalaninecompletelyabolishestheinter-actionbetweenfactorIXandfactorVIII.However,thesinglemutationofAsn89toalaninehasonlyamarginaleffectonbinding.Thetwoconsecutiveglycineresiduesintheleft-handedhelixhavebeenimplicatedasprotein-stabilisingfactorsinthemerozoitesurfacepro-TheÞrstaminoacidinthehelixinproteinC(Asp101)participatesinanunusualhydrogenbondinginteractionwithanotheracidicresidue(Glu85).Mutationsofthreeresiduesintheleft-handedhelix,namelyAsp101,Gly103andGly104,causeproteinCdeÞciencywhichmayleadtorecurrentvenousthrombosis,whichinturnmaycauseneonataldeath. Table2.Comparisonofsecondarystructureassignmentsofleft-handedhelicesbydifferentmethodsPDBcodeandchainIDType1bd0AHHHHHHHHHHHHHHTT(L)TGGGCHHHH-(R)1autLGGGGGGGGGGGGG(L)GGGGGGGG(L)1b9wAGGGGGGGGGGGGG(L)GGGGGGG-(L)1g2l_GGGGGGGGGGGGG(L)GGGGGGG-(L)1kliLGGGGGGGGGGGGG(L)GGGGGGG-(L)1n1iAGGGGGGGGGGGGG(L)GGGGGGG-(L)1ob1CGGGGGGGGGGGGG(L)GGGGGGGG(L)1rfn_GGGGGGGGGGGGG(L)GGGGGGGG(L)1pb5AGGGGGGGGGGGGG(L)GGGCÐ1kdgAGGGGGGGGGGGGG(L)GGGGGGGG(L)1hzmAHHHHHHHHHHHHH(L)TTTTHHHH(L)1gtxAHHHHHHHHHHHHH(L)HHHHHHHH(L)1qj5AGÐÐTTTT(L)HCCCÐ2gsaAGGGGGHHHTGGGT(L)GGGTÐ2oatAGHHHHGGGGGGGG(L)GGGGGGGG(R)1bnlAHHHHHHHHHHHHH(L)TTTTHHHH(L)1dy2AHHHHHHHHHHHHH(L)GGGTHHHH(L)1koeSHHHHHHHHHHHHH(L)TTTTHHHH(L)1bqbAHHHHHHHHHHHHH(L)GGGCHHHH(R)1npcEHHHHH/leftHHHHHHHH(L)GGGCHHHH(R)8tlnEHHHHH/leftHHHHHHHH(L)GGGCHHHH(R)1j9qAGGGGGGGGGGGGG(L)GGGCGGGG(R)1nifAGGGGGGGGGGGGG(L)GGGGGGGG(R)1oe1AGGGGGGGGGGGGG(L)GGGGGGGG(R)1ptmAGGGGGÐGGGGGGGGGGGG(L)GGGGGGGGGGGG(L)1ak0EHHHH-HHHTHHHT(L)TGGGHHHH(L)1mzr_GÐTTTTTTTT(L)TTTTÐ1h21AGGGGÐGGTTTGGTTT(L)GGTTTGÐ(R)1hxxAGHHHHHHHHHHHH(L)TGGGÐ1jv1AGGGGGGGGGGGGG(L)GGGGGGGG(L)1kwsAHHHHHHHHHHHHH(L)TTTTHHHH(L)Thefour-characterPDBcodeislisted,followedbythechainidentiÞer(whereanunderscoresigniÞesablankchainidentiÞer).TheentriesarelistedinthesameorderasinTable1Thetypeoftheleft-handedhelicesislistedasassignedmanuallybasedonhydrogen-bondingpatterns(H-helix;GSecondarystructureassignmentfortheresiduesineachleft-handedhelixintheirparentPDBÞle.Ð,nosecondarystructuretypewasassigned,His-helix,G3-helix,Tturn,andSbend.Theassignedhandedness(ifreported)islistedinparenthesesasRforright-handedandLforleft-handed.Secondarystructureassignmentfortheresiduesineachleft-handedhelixaccordingtotheprogramslisted. Left-handedHelices residueisthesubstrate-bindingresidueinthisfamilyofproteinsaswell.EndostatinsLeft-handed-helicalsegments(withidenticalsequences)wereidentiÞedinthreedifferentendo-statins(PDBcodes1bnl,1dy2,1koe).Aconservedcysteineresidueinthesehelicesparticipatesinadisulphidebridgethatiscrucialforthestabilityoftheseproteins.ProteinsfrompeptidasefamilyM14Aleft-handed-helixwasfoundinthreerelatedproteases,namely,inthermolysin(PDBcode8tln),aurolysin(1bqb)andneutralprotease(1npc),andthesequenceofthehelixisidenticalinallthree.Theside-chainoftheÞrstaminoacidinthehelix,aspartate,formsahydrogenbondwithahistidinethatcoordinatesthezincionintheactivesite.Thereisalsoahydrogenbondbetweentheback-boneofthisaspartateandthezinc-coordinatinghistidine,whichfurtherunderscorestheimport-anceofthehelicalsegmentforthecatalyticmechanismoftheseproteases.NitratereductaseNitratereductasesmediatethesecondreactionintheN-Þxationpathway,thereductionofnitratetoNO.Threebacterialnitratereductaseswerefoundtocontainaleft-handed3-helix(PDBcodes1j9q,1nifand1oe1).Thesecondaminoacidinthehelixisaleucineresiduethatispartoftheactivesitepocketwhereitalsohydrogenbondstoanorderedwatermolecule.4-Hydroxythreonine-4-phosphatedehydrogenase(PdxA)PdxA(PDBcode1ptm)isahomodimericenzymeinthepathwayleadingtopyridoxal5-phosphate,anessentialcofactorformanyenzymesinaminoacidmetabolism.PdxAcontainsthelongestleft-handedhelixencounteredinthissurvey,asix-residuelong3-helixthatislocatedinthedimerinterface.Ahistidineresidueinthehelix(His211),togetherwithtwohistidineresiduesfromtheothermonomer,coordinatesaZnthatisrequiredforcatalyticactivityoftheenzyme(Figure2thesubstrate4-hydroxy-l-threonine-phosphate(HTP)hasenteredtheprotein,ahydrogenbondisformedbetweentheOoxygenatomofHTPandtheNatomofHis211.Thus,thishistidineresidueparticipatesinbothmetal-bindingandsubstrate-binding.Itisconservedinall74knownsequencesofthePdxAfamilyNucleasePNucleaseP(PDBcode1ak0)isaphospo-diesterasethatdegradesbothsingle-strandedRNAandDNA.Theenzymecontainsashortleft-handed-helix(Ala131toGly134)thatformsapartoftheactivesitecleftoftheenzyme.Asn135,immediatelyfollowingthehelix,formsahydrogenbondwiththesubstrate.Thisenzymecontainszincions,andoneofthemiscoordinatedbyHis126,whichliesÞveresiduesupstreamoftheleft-handed2,5-Diketo--gluconicacidreductase(DkgA)DkgA(PDBcode1mzr)isanenzymeinthemetabolicpathwaytovitaminC.Ashort3-helixwasidentiÞedinvolvingaminoacids191to194.Leu190ofthisenzymecoordinatesthephosphategroupofthecofactorNADPH(S.Jeudy,personalcommunication).RemainingcasesFortheremainingproteinsthatcontainaleft-handedhelix(PDBcodes1hxx,1kws,1jv1and1h21)noindicationcouldbefoundastotheirpossiblestructuralorfunctionalroles.ConcludingremarksAnextensivesurveyofleft-handedhelicesinknownproteinstructureshasbeencarriedout.Theresultsshowthatleft-handedhelicesareraremotifsinproteinstructures(31casesin7284proteins).Aminoacidresiduesinthese31left-handedhelicesanglesbetween30and130betweenand100.TheserangesagreewellwithpreviousdeÞnitions.Mostoftheleft-handedheliceswereonlyfourresiduesinlength,andtheyoccurasboth-helicesand3-helices.Mostofthemcouldbeshowntoplayanimportantroleeitherforthestabilityorforthefunctionoftheprotein(e.g.determiningsubstratespeciÞcity,mediatingproteinÐproteininteractions,orbindingacofactorofanenzymaticreaction).Considerationofthehandednessofhelicescouldthereforehelppinpointfunctionallyinterestingpartsofthepro-tein.Thiscanbecomeespeciallyimportantasweenterthestructuralgenomicseraandtheneedforautomaticannotationofstructuresgrows,inpar-ticularforstructuresofproteinsofunknownfunction.Ourresultsalsoshowthatsecondarystructureassignmentisnotatrivialproblemandthatdifferentanswerscanbeobtainedforagivenpieceofstructurewhendifferentprogramsareused.Thesameistrueforthedesignationofhydrogenbonds,wherediscrepanciesbetweentheresultsofDSSP,DeepViewandHBPLUSwereobserved(datanotshown).TheUppsalaElectronDensityServerwasahelpfultooltoassesswhichhitsweregenuineandwhichwereartefactualleft-handedhelices.Ofthe56putativeleft-handedhelicesthatwereidentiÞed,33occurredincrystalstructuresandfor19oftheseelectrondensitymapswereavailablefromEDS. 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Left-handedHelices