Nature 兔朊蛋白的独特结构

1UniqueStructuralCharacteristicsoftheRabbitPrionProteinYiWen1,†,JunLi1,†,§,WenmingYao1,MinqianXiong1,JingHong1,YuPeng1,GengfuXiao2,andDonghaiLin1,3,*From1NMRLaboratory,ShanghaiInstituteofMateriaMedica,ChineseAcademyofSciences,Shanghai201203,and2StateKeyLaboratoryofVirology,CollegeofLifeScience,WuhanUniversity,Wuhan430072,and3KeyLaboratoryofChemicalBiologyofFujianProvince,CollegeofChemistryandChemicalEngineering,XiamenUniversity,Xiamen361005,China.Runninghead:Uniquerabbitprionprotein*Addresscorrespondenceto:DonghaiLin,ShanghaiInstituteofMateriaMedica,ChineseAcademyofSciences,555ZuChongzhiRoad,Shanghai201203,China.Tel/Faxnumber:[phone];E-mailaddress:[e-mail]ammalianspeciesthatappeartoberesistanttotransmissiblespongiformencephalopathies(TSEs)duetothestructuralcharacteristicsoftherabbitprionprotein(RaPrPC)itself.HerewedeterminedthesolutionstructuresoftherecombinantproteinRaPrPC-(91-228)anditsS173Nvariant,anddetectedthebackbonedynamicsoftheirstructuredC-terminaldomains-(121-228).IncontrasttomanyothermammalianPrPCs,loop165-172thatconnectsβ-sheet-2andα-helix-2iswell-definedinRaPrPC.Forthefirsttime,orderparametersS2areobtainedforresiduesinthisloopregion,indicatingthatloop165-172ofRaPrPCishighlyordered.Comparedwiththewild-typeRaPrPC,lesshydrogenbondsformintheS173Nvariant.TheNMRdynamicsanalysisrevealsadistinctincreaseinthestructuralflexibilityofloop165-172andhelix-3aftertheS173Nsubstitution,implyingthattheS173Nsubstitutiondisturbsthelong-rangeinteractionofloop165-172withhelix-3,whichfurtherleadstoamarkeddecreaseintheglobalconformationalstability.Significantly,RaPrPCpossessesauniquechargedistribution,carryingacontinuousareaofpositivechargesonthesurface,whichisdistinguishedfromotherPrPCs.TheS173NsubstitutioncausesvisiblechangesofthechargedistributionaroundtherecognitionsitesforthehypotheticalproteinX.Ourresultssuggestthattheorderedloop165-172anditsinteractionwithhelix-3,togetherwiththeuniquedistributionofsurfaceelectrostaticpotential,significantlycontributetotheuniquestructuralcharacteristicsofRaPrPC.Transmissiblespongiformencephalopathies(TSEs)orpriondiseases,whichincludescrapieinsheep,bovinespongiformencephalopathy(BSE)incattleandCreutzfeldt-Jakobdisease(CJD)inhumans,areanunusualgroupoffatalneurodegenerativedisordersthatcanbesporadic,inheritedoracquired.Theinfectiousagenthasbeenuniquelyidentifiedasthescrapieprionprotein(PrPSc),apathogenicisoformofthehost-encodedcellularprionprotein(PrPC)(1,2).PrPCandPrPScseemtopossessthesamecovalentstructurebutdiffersubstantiallyinconformation.PrPCismonomericandsoluble,sensitivetoproteolysiswithproteinaseK,whilePrPScishighlyinsolubleandreadilyformingproteinase-resistantaggregates.Bothcirculardichroism(CD)spectraandFouriertransforminfrared(FTIR)spectrashowthatPrPCispredominantlyα-helical,whereasPrPScpossessesaconsiderableamountofβ-sheetcontent(3-5).PrPCismostlyexpressedinthecentralnervoussystem.Itisahighlyconservedcellsurfaceglycoprotein,attachedtotheouterleafletofthecellmembraneviaaglycosylphosphatidylinositol(GPI)anchoratitsC-terminus.AlthoughthephysiologicalfunctionofPrPCremainsunknown,itshighaffinityforcopper(Ⅱ)indicatesthatitmayactasacoppertransportproteinorasuperoxidedismutase(6,7).PreviousstudieshaveindicatedthatPrPCmaybeinvolvedinpathwaysrelatedtocelladhesion,synapticintegrityandcellsignaling(8,9).Theconformationalconversionoftheprionprotein,fromPrPCtoPrPSc,hasacrucialroleinthepathogenesisofTSEs.Thus,knowledgeofthethree-dimensionalstructureoftheprionproteinisofgreatimportancetounderstandtheconformationalconversion.Solutionstructuresof(10-16).MaturePrPCconsistsofasinglepolypeptidechainofapproximately210aminoacids(residues23-231).TheN-terminus(residues23-120)isflexibleanddisordered,withahighlyconservedocta-repeatingsequencePHGGGWGQbetweenresidues60and91.TheC-terminus(residues121-231)isaglobularstructureddomainencompassingthreeα-helicesandtwoshortanti-parallelβ-strands,withadisulfidebondbridginghelices2and3.Loop165-172andhelix-3,socalledconformationalmarkers,arelocatedonthesurfaceofthestructuredC-terminaldomainandreflectthedifferencesamongspecies(17).RabbitsareoneofthefewmammalianspeciesthatappeartoberesistanttoTSEagents.SofarnosignsofTSEdiseaseshavebeenobservedinrabbitsafterinoculatingthemwiththeCJD,kuruorscrapieagentsisolatedfromeithermiceorsheep(18).BothrabbitPrPC(RaPrPC)andchimericrabbit-mousePrPCconstructsarenotconvertedtotheproteinase-resistantforminscrapie-infectedmouseneuroblastomacells(Sc+-MNBcells)whichaccumulatemousePrPSc(mPrPSc)(19).TheseexperimentssuggestthattheinabilityoftheconformationalconversionforRaPrPC