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Waksman, Gabriel and Orlova, Elena (2014) Structural organisation of the
type IV secretion systems. Current Opinion in Microbiology 17 , pp. 24-31.
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Structural
organisation
of
the
type
IV
secretion
systems
§Gabriel
Waksman
and
Elena
V
Orlova
TypeIVsecretion(T4S)systemsarelargedynamic
nanomachinesthattransportDNAsand/orproteinsthroughthe membranesofbacteria.Becauseoftheircomplexityand multi-proteinorganisation,T4Ssystemshavebeenextremely challengingtostudystructurally.Howeverinthepastfiveyears significantmilestoneshavebeenachievedbyX-ray
crystallographyandcryo-electronmicroscopy.Thisreview describessomeofthemorerecentadvances:thestructuresof someoftheproteincomponentsoftheT4Ssystemsandthe completecorecomplexstructurethatwasdeterminedusing electronmicroscopy.
Addresses
InstituteofStructuralandMolecularBiology,UCLandBirkbeckCollege, MaletStreet,WC1E7HXLondon,UK
Corresponding authors: Waksman, Gabriel
(g.waksman@mail.cryst.bbk.ac.uk)andOrlova,ElenaV (e.orlova@mail.cryst.bbk.ac.uk)
CurrentOpinioninMicrobiology2014,17:24–31
ThisreviewcomesfromathemedissueonHost–microbe interac-tions:bacteria
EditedbyOliviaSteele-MortimerandAgatheSubtil
1369-5274/$ –see front matter, #2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.mib.2013.11.001
Introduction
Nearly all types of bacteria (Gram-negative, Gram-positive,cell wall-less bacteria) and some archaeahave evolvedsecretionsystemsessentialforboththeirsurvival andvirulence[1].SecretionsystemstransportDNAand effector molecules such as enzymes or toxins from the bacterialinteriortoitsexterior.Understandingthe prin-ciplesofactionofthesenanomachineshasbroadclinical significancenotonlyduetodeliveryofbacterialtoxinsor effectorproteinsstraightintotargetedhostcells,butalso forthedirectinvolvementintherapidhorizontalspread ofantibioticresistancegenesamongthemicrobial popu-lation.
Secretion systems found in bacteria are classified cur-rently into seven major classes, among which type IV secretion(T4S)systemsformthemostfunctionally ver-satile class [2–7]. The variety of substrates and their nature (single proteins, protein complexes, DNA and nucleoproteincomplexes) secreted bytheT4S systems indeedsingleoutthisclassofsecretionsystemsfromthe others.The T4S systems areclassified into three func-tional groups. The first group mediates the transfer of DNAfromonebacterialcelltoanotherinaprocesscalled conjugation that plays an important role in bacterial genomeplasticity and diversity. Another group of T4S systemsmediates the translocation of proteins, ranging fromsmallproteineffectorsto largeproteincomplexes. Pathogenic Gram-negative bacteria such as Helicobacter pylori,BrucellasuisandLegionellapneumophilausetheT4S systemtoinjectvirulenceproteinsintomammalianhost cells[8–10]andBordetellapertussisusetheT4Ssystemsto secretepertussis toxinintotheextracellular milieu[11]. Thethirdgroup mediates DNArelease anduptake. H. pylori and Neisseria gonorrhoeae typify bacteria with this typeofT4S systems[13].T4S systemsofthefirstclass representanenormouspublic-healthproblemastheyare involved in the rapid dissemination of antibiotic-resist-ancegenes andother virulencetraitsamong pathogens. While the fact that DNA can move from one cell to another has been established a long time ago [12,15], themechanismofsecretionwaspoorlyunderstoodsince no structural information was available until very recently.
The T4S systemsare evolutionarily related:all nucleo-proteinandproteincomplexes aresecreted bytheT4S systems in an ATP-dependent process using a specific channelthroughacellenvelope[14].The T4Ssystems shareanumberofmaincomponentsanditseemsthatthe secretion processof all typesof substrates hascommon features[16].Inthis review we describethe latest pro-gressinstructuralstudiesoftheT4Ssystemcomponents andtheirmajorcomplexes.
Overall
organisation
of
T4S
systems
IthasbeenshownthattheT4Ssystemsspantheentire bacterialcellenvelope,creatingatranslocationchannel for varioussubstrates [1]. Almost all T4S systems of Gram-negativebacteriaminimallyconsistof12proteins namedVirB1toVirB11andVirD4(basedonthe nomen-clatureusedforAgrobacterium tumefaciensT4Ssystem) thatformamulti-proteinenvelope-spanningtransport apparatus(Figure1).Eachproteinispresentinmultiple copies.Thetwelvecomponentsareorganisedinthree major subcomplexes. A cytoplasmic-inner membrane (IM)subcomplexiscomposedofthreeATPases(VirB4,
§
VirB11andVirD4)andtheVirB3,VirB6andpartsofthe VirB8andVirB10proteins.TheATPasespowersystem assembly and substrate translocation [17–21]. VirB3, VirB6, andVirB8 arelikely to anchorVirB4 (and also perhapsVirB11)totheIM,participateintheIM chan-nel,andprovidealinktothecorecomplex(CC).The CC is composed of three proteins: VirB7, VirB9 and VirB10, and is a large central structure of the T4S system, that serves as scaffolding for the rest of the T4S system components.This complex formsa trans membrane spanning pore inserted both in the outer membrane (OM) and IM of Gram-negative bacteria and participates actively in T4S substrate transfer throughthebacterialenvelope[1,22,23,24].Thethird subcomplexisformedbytheVirB2andVirB5proteins that makethe extracellular pilusthat isimportant for directcontactwiththerecipientcellsurfaceandmayact asaconduitdeliveringthesubstratetotherecipientcell (Figure 1). While there are some deviations in the composition ofthese subcomplexesbetween different typesofT4Ssystemstheyarelikelyconservedintheir overallorganisation[1].
Advances
in
the
structural
analysis
of
T4S
systems
The complexity and dynamicsof the T4S systemsand theirmultiproteinorganisationrepresentserious difficul-tiesforthestructuralanalysisofT4Ssystemsoreventheir components.Onlyduringthelastdecadestructural infor-mation has emerged for the VirB/D system from A. tumefaciens and theclosely related systems from Escher-ichiacoli encodedbytheconjugative plasmidsF,R388, pKM101andRP4[25,26].X-raycrystallographicstudies of the T4S system have been performed on soluble proteincomponents(VirB11,thecytoplasmicregionsof VirD4and VirB4,theperiplasmicdomainof VirB8,and VirB5) and on the OM-embedded region of the CC (Figure 2).
Structures
of
ATPases
VirD4homologuesareessentialT4SATPasesthatactas substratereceptorsattheentryoftheT4Sapparatusand mightpowersubstratetranslocationthroughtheIM[21]. Because of their role in delivering thesubstrate to the translocation channel, VirD4 proteins are termed StructureoftheT4SSsWaksmanandOrlova 25
Figure1
VirD4
OM
IM VirB7
VirB4
VirB9
VirB8
VirB3
VirB10
VirB10
VirB2
VirB6
VirB5
Periplasm
Substrate
Cell Membrane
VirB11
Cytoplasm External Medium
Current Opinion in Microbiology
‘couplingproteins’. The crystalstructure of thesoluble
50kDa cytoplasmic domain of TrwB (VirD4 homol-ogue)from the R388 conjugative plasmid (PDB 1GKI,
Figure2A)revealedahomohexamerinwhicheach sub-unithastwodistinctdomains:anall-alphadomainfacing thecytoplasmandanATP-bindingdomainlinkedtothe IMbytheN-terminalmembraneanchor[17].The struc-tureimpliesthatVirD4couldfunctionasarotarymotor thatassists ssDNA and/or proteintranslocationthrough
the IM. Another recent evidence of involvement of couplingproteins inengagement of substratesand acti-vation of the secretion system was demonstrated for DotL,anIMcomponentoftheDot/Icmsecretionsystem inL.pneumophila.DotLacts asanIM receptorfor sub-stratesandrequiresadaptorproteinsforthesecretionofa majorclassofsubstrates[27].The DotLproteinis ana-logous to VirD4 in A. tumefaciens, and TraB in E. coli pKM101plasmid [28].Asin all membersof the VirD4 Figure2
90°
VirB10
VirB7
VirB9
TraC/VirB5 TcpC
centD TcpC CtermD
B. suis A. tumefaciens TraM
TrwB/VirD4
CTD
(a)
(d)
(f)
(e)
(b) (c)
VirB4CTD B. suis H. pylori
CTD
NTD NTD
E. faecalis
C. perfringens
Current Opinion in Microbiology
Structures of T4S system components or domains. (a) VirD4: structure of the soluble domain of TrwB. One subunit of the hexamer is shown (coils are shownincyan,helicesinblue,andstrandsinred).(b)VirB4:C-terminaldomainofThermoanaerobacterpseudethanolicusVirB4;(c)VirB11:crystal structuresofB.suisVirB11andH.pyloriHP5025,CTD-Cterminaldomain,NTD-Nterminaldomain(showninreddashedovalonB.suisVirB11).Red arrowindicatestheshiftoftheNTDinH.pyloriVir11comparedtoB.suisVirB11.(d)VirB8:crystalstructureoftheperiplasmicdomain(C-terminal domain) of VirB8 from B.suis(left panel), A.tumefaciens(middle panel), TraM214–322protein (right panel); the central and C-terminal domains of the
TcpC99–359structure are shown in the bottom panel. (e) VirB5: crystal structure of TraC encoded by the E.coliconjugative plasmid pKM101. (f) Crystal
structureoftheCC’sO-layercomposedofVirB7(indarkred),VirB9CT(ingreen)andVirB10CT(blue).Allstructuresareshownintheribbon
family, DotL also contains a Walker A motif for ATP hydrolysis andis believedto provideenergy toactively drive theexportofsubstratesacrosstheinner andouter membranesof thebacterialcellwall[27].
TheVirB4ATPaseoftheT4Ssystemisalsoessentialfor the assembly of the system and substrate transfer. Recently thecrystalstructureoftheC-terminaldomain ofThermoanaerobacterpseudethanolicusVirB4(PDBs4AG6 and 4AG5) has been obtained [20]. This structure is similar to VirD4, althoughit sharesonly 12% sequence identity. The VirB4domain crystallizes as amonomer, butthefull-lengthproteinisobservedinmonomer–dimer equilibrium,evenin thepresence of nucleotidesand/or DNAs.TheVirB4proteinssharefourcommonsequence motifsincludingtheconsensusWalkerAandB nucleo-side triphosphate-binding motifs (Figure 2B). A VirB4 topologymodelsuggestspossibleperiplasmicloops,one neartheNterminusandasecondontheN-terminalside of the Walker A motif [20]. The cellular localization, topology and oligomerization state of VirB4 remain unclear: different studies have reported dimeric and higher oligomeric (tetrameric and hexameric) forms of theprotein[29–32].ItwasshownthatVirB4canbebound throughitsN-terminaldomaintothecore’sVirB9protein [20].VirB4isinvolvedinearlierstepsofsubstrate engage-ment, and makes a number of additional interactions, notably withVirB3andVirB8[33–35].
VirB11isaperipheralmembraneproteinandbelongstoa family of ATPasestermed ‘traffic ATPases’ [19,18,36]. Recent studies have shown that conformation of VirB11(TrwD) canbe regulatedby magnesium,that at physiologicalconcentrationsinducesamorerigid confor-mation of the enzyme hence slowing down its activity [37]. The crystal structure of theADP-bound H. pylori VirB11 homologue (PDB 2PT7) revealed that each monomer consists of two domains formed by the N-terminal and C-terminal halves of the protein [19]. The nucleotide-binding siteisattheinterfacebetween thetwodomains.Inthehexamer,theN-terminaldomain andC-terminaldomainformtwoseparateringsdefininga chamberof50A˚ .ComparisonofthestructuresofB.suis (2GZA) and H. pyloriVirB11 (PDB2PT7)showed that theB.suisVirB11monomerdifferssignificantlyfromthat of H. pylori by a N-terminal domain swap that greatly modifies the nucleotide-binding site and the interface betweensubunits(Figure2C)[18].
Structures
of
VirB5
and
VirB8
proteins
VirB8 homologues are essential for substrate transfer throughtheIM.Ithasbeenproposedthattheyarepart of the inner-membrane pore and interact with VirB3, VirB4,VirB5andVirB6[35,38,39].TheN-terminal extre-mity of the structure is connected to a transmembrane segmentinsertedintheIM.Astructureoftheperiplasmic domain(C-terminaldomain)ofVirB8hasbeenobtained
for B. suisand A.tumefaciens (PDBs 2BHM and 2CC3) [38–40].StructuralcomparisonofB.suisandA.tumefaciens VirB8 confirms that the monomers have a similar fold (Figure 2D). Itcomprises an extendedb-sheetflanked with a-helices. Bothproteins exist as homodimers,and aminoacidsatthedimerinterfacearecriticalforprotein function. Smith et al. have found that E115 and K182 interact withinhibitorswhichapparently induce confor-mational changesthatpreventsVirB8-VirB8and VirB8-VirB10interactions[41].Recentlyitwasshownthatthe C-terminalcomponentofthetransferTraMprotein(214– 322aa,PDB4EC6)from theEnteroccocusfaecalis conju-gative plasmid pIP501 and the conjugation protein TcpC99–359 (PDB 3UB1) from Clostridium perfringens (Gram-positive)plasmidpCW3arestructurally homolo-goustotheperiplasmicregionofVirB8[42,43].TraM and TcpC are found localized to the cell membrane. Despite the absence of sequence-based similarity, the crystalstructuresofbothproteinsdisplayfoldssimilarto the T4S system VirB8 proteins from B. suis and A. tumefaciens. Surprisingly, the TcpC99–359 structure com-prisestwoVirB8-likedomainsseparatedbyalinker.The C-terminal domain iscriticalfor interactions withother conjugationproteins(Figure2D).Thestructure, molecu-lar dynamics, and cross-linking studies indicate that TraM isactiveas atrimer.The oligomerizationstateof TcpC isnotknown.
The crystal structure of TraC, a VirB5 homologue encoded by the E. coli conjugative plasmid pKM101 (PDB1R8I,Figure2E),hasbeen obtainedat3A˚ resol-ution[44].TheVirB5structurecomprisesa3-helix bun-dleflankedbyasmallerglobulardomain.Togetherwith the mainpilin VirB2, VirB5homologues form theT4S pilus, possiblyacting asadhesins.
Structures
of
the
CCs
Themostadvancedstructuralworkdescribedtodateisthe X-raycrystalstructureforthepartoftheCCthatlocatesin andneartheOM.Thispart,termedthe‘O-layer’,forms theOMporeoftheT4Ssystem(PDB3JQO)[45].Itis composedofthreeproteins/domains:theTraF/VirB10 C-terminal domain (TraF/VirB10CT), the TraO/VirB9 C-terminaldomain(TraO/VirB9CT),andTraN/VirB7,each in 14 copies. The structure has unveiled a surprisingly intricatenetworkofinteractionsbetweenthesethreemajor componentsoftheCC(Figure2F).VirB7seemstoworkas astaplerthatfastensVirB9andVirB10together;theinner surfaceoftheOMchannelislinedbyVirB10.Adiameterof the a-helical pore in thecrystal structureis 32A˚ that could accommodate the passage of DNA and unfolded proteinsubstrates.
structureoftheentireCC(Figure3).Thefirststructureof thecompleteCC(Figure3A)wassolvedbycryo-electron microscopy(cryo-EM)at15A˚ resolutionusingimagesof frozen samples [25]. The overall dimensions of the complexare185A˚ inbothheightanddiameter.Itconsists oftwolayers,termedOandI.TheO-layerhasaninner chamberthatisopento theextracellularmediathrough anopening of 10–20A˚ in thecap. Thecomplex hasan innerchannelthatspansthestructurefromtheOMtothe IM.Lateron,theCCstructurewasrefinedtoaresolution of12.4A˚ (Figure3B)whichrevealedmoredetailsofthe inner central channel [46]. In this higher resolution structure, the I-layer has a diameter of 50–55A˚ and itsinnerwallisformedby14columnsofdensitywitha diameterof8A˚ .ThecolumnsprojectfromtheO-layer of the CC to thebottom of theI-layer where they are connectedtothebaseofthecomplexdefininganopening of 55A˚ in diameter on the cytoplasmic side of the complex.
Digestion of the CC with elastase produced a stable truncated complex, termed the ‘CCelastase’ complex. ThesequencingofthebandsinSDS-PAGEgelsconfirms thatthecomplexismadeoffull-lengthTraN/VirB7and
TraO/VirB9,andTraF/VirB10CT(Figure3C).Astructure of CCelastase wasobtained ataresolution of 8.5A˚ using cryo-EMandsingle particleanalysis(Figure3D)[46]. Comparison of full length CC and CCelastase demon-strated considerable differences in the I-layer region. Inthefull-lengthCC,theI-layercomprisesthe N-term-inaldomainsofVirB9(TraF/VirB9NT)andVirB10(TraF/ VirB10NT)and isinserted in theIM via theVirB10 N-terminaltrans-membranesegment.TheremovalofTraF/ VirB10NTbyelastaseresultsinanincreaseoftheopening onthecytoplasmicside(65A˚ comparedto55A˚ inthe CC)andashorteningof theI-layer(Figure3D).
IntheCCelastasecomplex,thecolumnswithinthecentral channel are absent (Figure 3E). Therefore the inner columns of the CC I-layer were assigned to TraF/ VirB10NT(Figure4A).ThemajorpartofTraF/VirB10NT wasshowntobeunstructured;theonlysecondary struc-tural elements that were reliably identified were three short helical elements: the helix corresponding to the inner-membrane spanning region (aA: K40AF-LVF53) and two other a-helical regions (aB: A107RA-QAA113; aC: P138EE-QRR146). Their length is compatible with thediameterandthelengthoftheobservedcolumnsin Figure3
TM SP
CTD CTD
B9BD
1 48
24 294
161 386
CCelastase CCelastase
TraF/VirB10 TraO/VirB9 TraN/VirB7
O layer
I layer
Cap
Base
Full-length
CC
I-layer:
O-layer -VirB7 -VirB9CT
-VirB10CT
-VirB9NT
-VirB10NT (a)
(c) (d) (e)
(b)
Current Opinion in Microbiology
ElectronmicroscopyoftheT4Ssystem.(a)StructureoftheCCcomplexat15A˚;(b)structureoftheCCcomplexat12A˚;(c)schematicillustrationof the regions of TraN/VirB7, TraO/VirB9, and TraF/VirB10 present in the CCelastasecomplex. Domains corresponding to the VirB9 binding domain (B9BD),
the signal peptide (SP), the N-terminal trans-membrane (TM) helix and the C-terminal domains (CTD) are shown in darker colours; (d) Cryo-EM structureoftheCCelastasecomplex.(e)Cutawayviewofthesuperpositionofthedifferencemap(ingreen)betweenthefulllengthCCandCCelastaseand
theCCcryo-EMmap([46],andreferencesherein]).At theverybottomofthecomplexthesestretchesjoineach othertoformaringatthebaseoftheCC.Thisringmight correspond to the 14 trans-membrane segments (helix aA)associatedwithdetergentmicelles.VirB10islinked to the IM by the VirB10 N-terminal trans-membrane helix(30–50aa)characterisedwithtwotypesofputative dimerization motifs:aGxxxA(GA4) motifandtwo leu-cine(Leu1,Leu2)zippers.WhilemutationsintheLeu1 motifdisruptT-pilusbiogenesis,thisandothermutations intheGA4orLeu2motifdonotabolishsubstratetransfer [47].
ThemodellingoftheatomicstructureofTraO/VirB9NT (residues 24–135) and the examination of its possible dockingintoEMmapshelpedlocatethisdomainwithin the structure. The results suggest that TraO/VirB9NT adopts a beta-sandwich fold (Figure 4B). Fitting of models for TraO/VirB9NT, and labelling experiments with nanobodiestargeted against TraO/VirB9NT are all pointing towards the conclusion that TraO/VirB9NT is locatedattheoutsidesurfaceof theI-layer.
Conclusions
During the last decade structural analysis has made a greatimpact onourunderstandingof howT4S systems are organisedandfunction.
TheEMstructureshavegreatlyalteredourconceptionof the entire T4S system architecture and its possible mechanism of action. It was found that the CC is an
essential structural portion of T4S systems. It is self-assemblingandthereforeislikelytobeassembledfirst. TheCCformstheIMchannelandisamajorcomponent of the OM pore. It provides the central scaffold for assemblyofallothercomponents.Structuralcomparison of the complete and truncated CCs demonstrate that TraF/VirB10spanstheentirecellenvelope.This obser-vation together with fitting of crystal and modelled structuresintoEMmapsoflargecomplexesisof particu-larinterestsinceitprovidestheexplanationhowVirB10 isabletosenseconformationalchangesinducedbyATP bindingtoorATPhydrolysisbytheT4SsystemATPases situatedattheT4Schannel’sentranceonitscytoplasmic side [23]. VirB10 respondsto thesechangesby under-going itsownstructuralmodifications thatare required forsubstratetransfer.Cascalesetal.havedemonstrated thatDNAbindingtoVirD4andVirB11stimulatesATP binding/hydrolysis, which in turn activates VirB10 through a structural transition [48]. Segure and co-authorshave shownthatthetransmembranedomainof TrwB/VirD4 is needed for the interaction with the transmembrane domain of TrwB/VirB10 and for R388 conjugativetransfer.Theremovaloffirst64aminoacids in TrwB/VirB10 reduced conjugation by six orders of magnitude[47].ThusproteinsanaloguestoVirB10may serve as a signal transmitter throughout the secretion complex[1,21,23,25,47,48,49].
The next mission in T4S studies is to establish the organisation and structure of even larger complexes of T4Ssystemcomponents.Forexample,itisimportantto StructureoftheT4SSsWaksmanandOrlova 29
Figure4
24 aa
αC
αB
αA
N-terminus
OM
IM
(a) (b)
O lay
er
I lay
e
r
Current Opinion in Microbiology
Schematicmodelforthefulllengthorganisationofthecorecomplex.(a)FourTraF/VirB10CTsubunitsofthe14-merpresentintheO-layeratomic
structureareshown.Onesubunitishighlightedinblue.Thedensityofonesubunitcolumninthedifferencemap(inlightgreen)isshownwiththe tentativedockingofthethreeTraF/VirB10NTa-helicalregions.ThissubunitislocatedimmediatelybelowTraF/VirB10CTshownindarkblue.The
completeourunderstandingoftheIMchannelby deter-miningthestructureofVirB6andVirB8boundtotheCC. Ultimately, the structure of the entire translocation machinewillneedto bedetermined.Arelatedquestion ishowsubstratetransfertakesplace.Thistaskrepresents atremendouschallengethatcanonlybemetby imple-menting novel biochemical and genetic approaches resultingin trapping T4Ssystems atdifferentstages of substrate transfer. The elucidationof T4S mechanisms will thus require joint efforts from biochemistry and structuralbiology.Thederivedknowledgemightbeused tofindwaysofinhibitingthesesystemstocombat infec-tionsandcontrolthespreadofantibioticresistancegenes.
Acknowledgements
TheworkontheCCandthe‘‘CCelastase’’complexoftheT4Ssystemwas fundedbygrant082227fromtheWellcomeTrusttoGWandK012401from MRCtoEVOandGW.TheauthorswouldalsoliketothanktheWellcome TrustfortheEMequipment.
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recommended
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