数据仓库数据挖掘chap2.

ACCEPTEDMANUSCRIPTcholesterol-dependentcytolysinStepwisevisualizationofmembraneporeformationbysuilysin,abacterialADunstone,PeterWAndrew,RanaLonnen,MayaTopf,HelenRSaibil,BartWHoogenboomPandurangan,NasrinJahan,MafaldaPiresDamaso,DinoOsmanovic,CyrilFReboul,MichelleCarlLeung,NatalyaVDudkina,NatalyaLukoyanova,AdrianWHodel,IreneFarabella,ArunP:Citeas:eLife2014;10.7554/eLife.04247Published:2December2014Accepted:24November2014Received:5August2014editing,andproofing.formattedHTML,PDF,andXMLversionswillbemadeavailableaftertechnicalprocessing,ThisPDFistheversionofthearticlethatwasacceptedforpublicationafterpeerreview.Fullyelife.elifesciences.orgatSignupforalertsStaycurrentonthelatestinlifescienceandbiomedicalresearchfromeLife.Membraneporeformationbysuilysin1Stepwisevisualizationofmembraneporeformationbysuilysin,1abacterialcholesterol-dependentcytolysin23CarlLeung1,7,NatalyaV.Dudkina2,7,NatalyaLukoyanova2,AdrianW.Hodel1,IreneFarabella2,ArunP.4Pandurangan2,NasrinJahan4,MafaldaPiresDamaso4,DinoOsmanović1,3,CyrilF.Reboul5,Michelle5A.Dunstone5,6,PeterW.Andrew4,RanaLonnen4,MayaTopf2,HelenR.Saibil2,8,*,andBartW.6Hoogenboom1,3,8,*781LondonCentreforNanotechnology,UniversityCollegeLondon,LondonWC1H0AH,UnitedKingdom92DepartmentofCrystallographyandInstituteofStructuralandMolecularBiology,BirkbeckCollege,10LondonWC1E7HX,UnitedKingdom113DepartmentofPhysicsandAstronomy,UniversityCollegeLondon,LondonWC1E6BT,United12Kingdom134DepartmentofInfection,Immunity,andInflammation,UniversityofLeicester,LeicesterLE19HN,14UnitedKingdom155DepartmentofBiochemistryandMolecularBiology,MonashUniversity,Clayton,Melbourne,Victoria163800,Australia176DepartmentofMicrobiology,MonashUniversity,Clayton,Melbourne,Victoria3800,Australia187Equalcontributions198Equalcontributions20*Correspondence:[e-mail],[e-mail]21Membraneporeformationbysuilysin2Abstract22Membraneattackcomplex/perforin/cholesterol-dependentcytolysin(MACPF/CDC)proteinsconstitutea23majorsuperfamilyofpore-formingproteinsthatactasbacterialvirulencefactorsandeffectorsin24immunedefence.Uponbindingtothemembrane,theyconvertfromthesolublemonomericformto25oligomeric,membrane-insertedpores.Usingreal-timeatomicforcemicroscopy(AFM),electron26microscopy(EM)andatomicstructurefitting,wehavemappedthestructureandassemblypathwaysof27abacterialCDCinunprecedenteddetailandaccuracy,focussingonsuilysinfromStreptococcussuis.28Weshowthatsuilysinassemblyisanoncooperativeprocessthatisterminatedbeforetheprotein29insertsintothemembrane.Theresultingring-shapedporesandkineticallytrappedarc-shaped30assembliesareallseentoperforatethemembrane,asalsovisiblebytheejectionofitslipids.31Membraneinsertionrequiresaconcertedconformationalchangeofthemonomericsubunits,witha32markedexpansioninporediameterduetolargechangesinsubunitstructureandpacking.3334Introduction35ThebacterialCDCsandubiquitousMACPFproteinsareexpressedassolublemonomersbutassemble36onmembranestoformlarge,oligomericpores.Theyformtwobranchesofthelargestsuperfamilyof37pore-formingproteins.ProteinsofthisMACPF/CDCsuperfamilyshareacommoncoretopologyofa38highlybentandtwisted-sheetflankedbytwoα-helicalregions,thoughlackinganydetectable39sequencehomologybetweenthetwobranches(Rosadoetal.,2008).CrystalstructuresofCDCsin40theirsoluble,monomericform(perfringolysin,Rossjohnetal.,1997;anthrolysin,Bourdeauetal.,2009;41suilysin,Xuetal.,2010;listeriolysin,Kösteretal.,2014)revealedextended,key-shapedmolecules.42Pore-formingdomains1and3(Figure1C)arelinkedbyalongthin-sheet(domain2)toan43immunoglobulinfolddomain(4)whichcanbindtothemembraneviaatryptophan-richloop.CDCsform44heterogeneousringsandarcs(Dangetal.,2005;Tilleyetal.,2005;Sonnenetal.,2014)on45cholesterol-richliposomesandlipidmonolayers(forexample,theCDCperfringolysinOhardlybindsto46membraneswith30%molarconcentrationofcholesterol,Johnsonetal.,2012).Extensivebiophysical47andmolecularanalysisofCDCsestablishedthat,onCDCbindingtothemembrane(Hotzeetal.,2012;48Ramachandranetal.,2004),α-helicalregionsindomain3unfurltoformtransmembrane-hairpins,49Membraneporeformationbysuilysin3denotedTMH1andTMH2(Shaturskyetal.,1999;Shepardetal.,1998).IftheTMHregionsare50trappedbyintroducingadisulphidebond(Hotzeetal.,2001),preporeoligomersareformedonthe51membranesurface.Cryo-EMandsingleparticleanalysisofliposome-boundCDCsledtolow-resolution523Dstructuresofpreporeandporeformsofpneumolysin,amajorvirulencefactorofStreptococcus53pneumoniae(Tilleyetal.,2005).Thesestructures,aswellasanAFMstudyofperfringolysin54(Czajkowskyetal.,2004),establishedthatthe11nmhighmoleculemustcollapsetoaheightof7nm55abovethemembraneinordertoinserttheTMHregions.Simplepseudo-atomicmodelswereobtained56byfittingdomains(brokenatplausiblehingepoints)intotheEMdensitymaps.Itwasproposedthatthe57long,thin-sheetdomain2collapsesafterthemoleculeopensuptoreleasetheTMHregions..58However,becauseoftheheterogeneityoftheoligomericassembliesandaggregationoftheliposomes59uponporeformation,resolutionhasbeenlimitedbythedifficultyofob