waterstablehost

SHORTCOMMUNICATIONSThreonineinCollagenTriple-helicalStructureNatthaJIRAVANICHANUN,1;2KazunoriMIZUNO,3HansPeterBA¨CHINGER,3andKenjiOKUYAMA1;y1DepartmentofMacromolecularScience,GraduateSchoolofScience,OsakaUniversity,Toyonaka560-0043,Japan2DepartmentofBiotechnologyandLifeScience,GraduateSchoolofEngineering,TokyoUniversityofAgricultureandTechnology,Koganei184-8588,Japan3DepartmentofBiochemistryandMolecularBiology,OregonHealth&ScienceUniversity,andShrinersHospitalforChildren,ResearchDepartment,Portland,Oregon97239,USA(ReceivedOctober4,2005;AcceptedDecember5,2005;PublishedApril15,2006)KEYWORDSThreonine/Collagen/Triple-helicalStructure/Host-guestPeptide/Side-chainConformation/HydrationPattern/[DOI10.
1295/polymj.
38.
400]Collagenisthemostabundantproteinsfoundintheextracellularmatrixofmulticellularanimals,andhasauniquetriple-helicalstructure,whichiscomposedofthethreepolypeptides.
Thethreechainsformaright-handedsupercoiledtriple-helix.
Eachpolypep-tidechainrequiresGlyateverythirdresidue,whichgenerates-Xaa-Yaa-Gly-repeatingsequence.
Theglycineresiduesineverythirdpositionarepackedinthecenterofthetriple-helix.
TheresiduesintheXaaandtheYaapositionsareexposedtothemolecu-larsurface.
HighcontentsofiminoacidsintheXaaandtheYaapositionsarerequiredtothestabilityofthestructure.
Collagenfamilyincludesmorethanthirtyproteinsinvertebrates.
1,2Similarcollagensandmuchmorediversecollagenproteinsarealsopresent-edthroughoutinvertebratesincludingafewgiantmoleculesfoundinthecuticlesofseveralwormspe-cies.
3,4Forexample,theRiftiapachyptilacuticlecol-lagenhasalowHypcontentbuttheThrcontentismuchhigherthanthosefoundinothercollagens.
5Themechanismofthestabilityofthecollagenhelixisstillunknown.
TheThrofthecollagenishighlygly-cosylated.
4Severalmodelpeptidesweresynthesizedtoanalyzeitsthermalstabilityandproperty.
5–9TheO-galactosylationofThrincreasesthethermalstability(thehelix-coiltransitiontemperature)ofAc-(Gly-Pro/4(R)Hyp-Thr)10-NH2peptides.
6TheCDexperimentsofAc-(Gly-4(R)Hyp-Yaa)10-NH2peptideswithvari-ousaminoacidsintheYaaposition(Thr,Ser,Val,Ala,andalloThr)suggestedthatthemethylgroup,hydroxylgroupandstereocongurationofThrareim-portantforthestability.
9ThemethylgroupofThrwashypothesizedtoshieldtheinter-chainhydrogenbondbetweentheamideofGlyandcarbonylofXaaresi-duesfromwatermoleculesbyenergy-minimizationmethod.
9Althoughseveralstudieshavechallengedtorationalizetheexperimentaldata,themechanismofthestabilityincuticlecollagenisstillambiguous.
InordertounderstandthestabilizationmechanismofThrintheYaaposition,weattemptedtocrystallizethepeptidesAc-(Gly-4(R)Hyp-Thr)10-NH2andH-(Gly-4(R)Hyp-Thr)10-OH.
Despitetheirabilitytoformatriple-helicalstructure,6wecouldnotsucceedintheformationofthesinglecrystalsofthesepeptidesyet.
Thehost-guestpeptidesystemisanalternativewaytogetsinglecrystalsofthepeptidewithinterest-ingsequence.
Therefore,4(R)Hyp-Thr-Glytripeptideunitwasinsertedintothestablehostpeptide(Pro-Pro-Gly)9.
10Thehost-guestpeptideH-(Pro-Pro-Gly)4-(4(R)Hyp-Thr-Gly)-(Pro-Pro-Gly)4-OH(OTG)con-tains4(R)Hyp-Thr-GlytripeptideunitthatisabundantintheRiftiapachyptilacuticlecollagen.
ThesinglecrystalanalysisoftheOTGpeptideprovidedtherstinsightintotheunique4(R)Hyp-Thr-Glytripeptideunitconformation.
Here,theThrconformationandtheobservedhydrationpatternsaroundThrresidueintriple-helicalstructurewererevealed.
EXPERIMENTALCrystallizationwasperformedbyhanging-dropdif-fusionmethodat4C.
Samplesolutionwaspreparedatconcentrated10mgmL1.
Reservoirsolutioncon-tained0.
1MHepesbuerpH7.
5and23%(w/v)PEG1000.
Dropmixturemadeupofsamplesolution2mlandreservoirsolution2ml.
Rod-likecrystalsap-pearedinabout3weeks.
Asinglecrystalwasmeas-uredat100KonthebeamlineBL6AatthePhotonFactoryinTsukuba.
IntensitydatawasprocessedbyCrystalClear.
11CrystalbelongstomonoclinicspaceyTowhomcorrespondenceshouldbeaddressed(Tel:+81-66-850-5455,Fax:+81-66-850-5455,E-mail:okuyamak@chem.
sci.
osaka-u.
ac.
jp).
400PolymerJournal,Vol.
38,No.
4,pp.
400–403(2006)groupP21withunitcellparametersa26:0,b26:5,c80:2A,90:4.
ThestructureofOTGwasdeterminedbymolecularreplacementmethodusing(Pro-Pro-Gly)9peptide(PDBcode1ITT)12asasearchmodel.
PositionalrenementwasperformedbyX-PLOR13andstructurerenementwascarriedoutbySHELX-L.
14RESULTSANDDISCUSSIONThrresiduesareatthecentraltripeptideunitofthemolecule.
Todescribeside-chainconformationofThr,1dihedralangleisdenedbyN-C-C-O1orN-C-C-C2.
Thedierentconformationsoftheside-chainasafunctionof1valuesof60,180,and60arereferredtogauche,trans,andgauche,respectively.
Thus,Thrside-chaininOTGstructure,theO1takesgaucheconformation,whereastheC2takestransconformationtoamidegroup(Figure1).
BoththeO1andtheC2aredirectedtowardadja-centchains.
ThiskindofThrside-chainconformationisthesameastwooutofthreeThrinT3-785pep-tide.
15Theaveragemain-chaindihedralangles(=)ofThrinthisstudyare61and145,whicharecon-sistentwiththosevaluesintheYaapositionofcolla-gen-likepeptides.
10,16InT3-785structure,15water-mediatedhydrogenbondwasreportedbetweentheThrOHgroupandtheGlycarbonylinthesamechainviaonewatermolecule.
ForThr,notonlytheabovewater-mediatedpattern,butalsodiversewater-medi-atedpatternsareobservedintheOTGstructureinFigure2a.
Intherstcase,twowatermoleculesmakehydrogenbondswiththeOHgroupofThr114andthegauche+NOγ1transCγ2Figure1.
gauche/transconformationofThrintheOTGstructure.
Gly1AOGly2BOGly112OThr314O1γHyp3AOThr114NHyp4BOδδδThr114O1Hyp5COHyp4BOThr114OThr214O1γγHyp5COPro317OHyp6AO(a)(b)Figure2.
(a)CentralregionoftheOTGmoleculeshowsthreecasesofwater-mediatedhydrogenbondsatOHgroupofThr.
Case1:Thr114O1connectsthroughtwowatermoleculestoGly112Ointhesamechain.
Case2:Thr114O1connectsthroughtwowatermole-culestoThr114Nwithinthesameresidue.
Case3:Thr314O1connectsthroughtwowatermoleculestoThr114OofadjacentchainandThr214O1connectsthroughtwowatermoleculestoPro317Oofadjacentchain.
Intheresiduename,therstdigitcorrespondstoachainnumberandthenexttwodigitscorrespondtoaresiduenumber.
(b)HydrationpatternsinvolvingOHgroupof4(R)Hypandcarbonylgroupsin(Pro-4(R)Hyp-Gly)11structure.
16Threechainsinamoleculeareshownbydierentshedding;light-,middle-anddark-gray.
Spheresarewatermolecules.
Intra-chainandinter-chainwater-mediatednetworksareshowninbrokenandsolidlines,respectively.
ThreonineinCollagenTriple-helicalStructurePolym.
J.
,Vol.
38,No.
4,2006401carbonylgroupofGly112inthesamechain(brokenlines).
Inthesecondcase,twowatermoleculesinter-actbetweentheOHandtheamidegroupswithinthesameThr114residue(brokenlines).
Andthethirdcaseoccursattwopositions;oneistwowatermole-culesarelinkedbetweentheOHgroupofThr314andthecarbonylgroupofThr114ofneighboringchainandanotheristhesimilarpatternbetweentheOHgroupofThr214andthecarbonylgroupofPro317(solidlines).
Theaveragehydrogenbonddis-tanceinthesethreewater-mediatedpatternsis2.
95A.
Thehydrationpatterninthesecondcasecouldnotbefoundat4(R)Hypduetothelackofhydrogenattheamidegroup.
However,hydrationpatternsintherstandthethirdcases,whichareinter-andintra-chainhydrogenbondnetworks,aregenerallyobservedat4(R)HypintheYaapositioninthepeptidesincludingPro-4(R)Hyp-Glytripeptideunit.
16,17Theseinter-andintra-chainhydrationnetworksoccurrepeatedlyalongthetriple-helicalmolecule,forexample,in(Pro-4(R)Hyp-Gly)11structure16asshowninFigure2b.
Theyarethedominantfeatureintherepetitivepat-ternsof4(R)HypinpeptideshavingPro-4(R)Hyp-Glyrepeatingsequence.
16,17Thus,thisresultindicatesthattheOHgroupofThrintheYaapositionpartici-patesinwater-mediatedinter-andintra-chainhydro-genbondsinthesimilarwaytotheOHgroupof4(R)Hyp.
Moreover,thepositionoftheOHgroupofThrislocatedclosetothatof4(R)HypwhenThrintheOTGstructureissuperimposedoverthe4(R)Hypinthe(Pro-4(R)Hyp-Gly)11structure(Figure3).
Thedistancebetweenbothhydroxyloxygenatomsisabout1A.
ThecloselocationoftheOHgroupsofThrto4(R)Hypcouldcontributetothesimilarforma-tionofwater-mediatedhydrogenbonds.
Therefore,theOTGstructuredemonstratesthatThrcouldactlike4(R)HypatOHgroupside-chaintomakesimilarwater-mediatednetworks.
ThethermalstabilityofAc-(Gly-4(R)Hyp-Yaa)10-NH2peptidescontainingtheThrishigherthanthosecontainingtheSer,alloThr,AlaandVal,9whichsuggeststheimportanceoftheside-chainconformationinthetriple-helicalstructure.
SofaronlytheT3-78515peptidehasareportedstructureofThrinatriple-helicalstructure.
TheX-raydeterminationoftheOTGpeptideprovidesinsightintodetailedstructureoffrequentlyobservedresiduesinRiftiapachytilacuticlecollagen.
Althoughthestabi-lizationmechanismoftheOTGpeptideisnotclearlyunderstood,thenestructureoftheOTGpeptidepro-videsvaluableinformationofThrconformationin-cludingdiversityofwater-mediatedhydrogenbondsaroundThrinthetriple-helicalstructure.
Interestingly,theobservedhydrationpatternsofThraresimilartothoseof4(R)Hypandmoreover,OHgroupside-chaincharacteristicofThrand4(R)Hypissimilaraswell.
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