Coiled-coils in type III secretion systems: structural flexibility, disorder and biological...

Microreview

Coiled-coils in type III secretion systems: structuralflexibility, disorder and biological implications

Anastasia D. Gazi,1,2† Spyridoula N. Charova,2†

Nicholas J. Panopoulos2 and Michael Kokkinidis1,2*1Institute of Molecular Biology and Biotechnology,Foundation of Research and Technology and2Department of Biology, University of Crete, VasilikaVouton, GR 71409, PO Box 2208, Heraklion, Crete,Greece.

Summary

Recent structural studies and analyses of microbialgenomes have consolidated the understanding ofthe structural and functional versatility of coiled-coil domains in proteins from bacterial type IIIsecretion systems (T3SS). Such domains consist oftwo or more a-helices forming a bundle structure.The occurrence of coiled-coils in T3SS is consider-ably higher than the average predicted occurrencein prokaryotic proteomes. T3SS proteins compris-ing coiled-coil domains are frequently character-ized by an increased structural flexibility, whichmay vary from localized structural disorder to theestablishment of molten globule-like state. Thepropensity for coiled-coil formation and structuraldisorder are frequently essential requirements forvarious T3SS functions, including the establish-ment of protein–protein interaction networks andthe polymerization of extracellular components ofT3SS appendages. Possible correlations betweenthe frequently observed N-terminal structural dis-order of effectors and the T3SS secretion signal arediscussed. The results for T3SS are also comparedwith other Gram-negative secretory systems.

Introduction

The type III secretion system (T3SS) is an essentialmediator of interactions between Gram-negative bacteria

and eukaryotes (Hueck, 1998; Cascales and Christie,2003; Tampakaki et al., 2004; Troisfontaines and Corne-lis, 2005). This pathway possesses dual roles: in initialstages of the T3SS assembly it contributes significantly toprotein secretion to the extracellular space, while efficientdelivery of bacterial proteins (effectors) to the eukaryoticcells takes place when the system is fully developed(translocation).

Although the T3SS apparatus is conserved in pro-karyotic pathogens across the plant/animal phyllogeneticdivide, the secreted proteins differ considerably and thediseases range from bubonic plague and septicaemia inhumans to localized lesions, systemic wilting and blightsin plants.

The T3SS are of two general types: flagellar andnon-flagellar. The latter have evolved into seven familieswhich are thought to have been exchanged amongGram-negative proteobacteria by horizontal gene transfer(Alfano and Collmer, 2004; Troisfontaines and Cornelis,2005; Abramovitch et al., 2006; Zhang et al., 2006). A setof nine proteins widely conserved among T3SS forms theinjectisome core. Sequence and structural homologiesbetween core proteins span the divide between flagellarand non-flagellar systems, as evidenced by the structuralsimilarities of the homologous domains HrcQB (Tampakakiet al., 2004) and FliN (Brown et al., 2005), the T3SSATPase EscN (Zarivach et al., 2007) and the flagellarATPase FliI (Imada et al., 2007). On the other hand, con-siderable sequence and structural variation exists amongother T3SS proteins and effectors (Tampakaki et al.,2004). Despite this diversity, however, T3SS proteinsreveal an usual propensity for formation of a recurrenttertiary motif, the coiled-coil (Delahay and Frankel, 2002;Pallen et al., 2005; Zhang et al., 2006) which suggeststhat it may contribute to important functions.

In the following, the coiled-coil domains of T3SS pro-teins and their structural plasticity, a poorly understoodproperty (Dyson and Wright, 2005), will be reviewed. Theresults will be also compared with other Gram-negativesecretory systems, i.e. (i) with type II secretion systems(T2SS), which utilize products of the sec genes to movepre-secretory proteins across the inner membrane fol-lowed by the processed mature protein being translocated

Received 14 October, 2008; revised 30 January, 2009; accepted2 February, 2009. *For correspondence. E-mail [email protected]; Tel. (+30) 2810394351; Fax (+30) 2810394351.†A.D.G. and S.N.C. have equally contributed to this work.

Cellular Microbiology (2009) 11(5), 719–729 doi:10.1111/j.1462-5822.2009.01297.xFirst published online 9 March 2009

© 2009 Blackwell Publishing Ltd

cellular microbiology

across the outer membrane (Pugsley et al., 1997) and (ii)with type IV secretion systems (T4SS) the functions ofwhich include (a) conjugal transfer of DNA by cell-to-cellcontact, (b) translocation of effectors to eukaryotic targetcells and (c) DNA uptake and release to the extracellularmilieu. T4SS comprise two major subgroups termedIVA (T4ASS) and IVB (T4BSS) respectively (Ding et al.,2003).

Coiled-coils and a-helical bundles

Coiled-coils consist of two to seven amphipathic a-heliceswinding around each other to form a supercoiled bundle(Fig. 1A–D). The helices may run parallel or antiparallel,and may form homo- or heterocomplexes (Grigoryan andKeating, 2008). Sequences of regular, left-hand twistedcoiled-coils are characterized by a seven-residue periodi-city (heptad repeat) of physicochemical properties ofside-chains. If the heptad positions are labelled a–g, thenpositions a and d are hydrophobic and form the core of thebundle (Fig. 1A-1D). Positions b, c, e, f and g are moresolvent-exposed and their amino acid preferences reflectconstraints which are specific to each type of helicalbundle (Lupas et al., 1991; Paliakasis and Kokkinidis,1992). Coiled-coil helices are distinguished from otheramphipathic helices by the periodicity of hydrophobic resi-dues (3.5 versus 3.65 residues per turn), the length (longversus short) and the packing interactions. However,there is a continuum of intermediate bundle structures;some proteins are induced to form coiled-coils upon asso-ciation with a binding partner (Lupas, 1996). Coiled-coilsare found for ~10% of all eukaryotic and 4–5% of allprokaryotic and archae proteins (Liu and Rost, 2001).Extended (more than 75 residues) coiled-coils occur fourtimes more frequently in the animal compared with theplant kingdom, and are rare in bacterial pathogens. Shortcoiled-coils occur with nearly equal frequency across allphyla, where they act as modular assembly domainsthrough homo- or heterodimer formation (Odgren et al.,1996).

Flexibility and intrinsic disorder

Proteins span a continuum from totally disordered to well-folded structures. Intrinsically disordered regions are very

common in proteomes and are frequently functionallycrucial, especially those involved in signalling, recogni-tion, regulation and self-assembly (Namba, 2001). Theextreme flexibility of intrinsically disordered proteins,whose structures may alter dramatically as they bind totheir cognate folded partner, has been suggested to rep-resent a strategy for optimizing the search and interactionwith their targets (Sugase et al., 2007). Intrinsically disor-dered proteins are substantially depleted in W,C,F,Y,V,L,N(order-promoting) and enriched in A,R,G,Q,S,P,E,K(disorder-promoting amino acids) (Dunker et al., 2002),from which it is predicted that a considerable number ofproteins (> 30% of eukaryotic proteins) have disorderedregions longer than 50 consecutive residues. Other pre-dictive methods, e.g. the FoldIndex (Prilusky et al., 2005),use average residue hydrophobicities and net charges.Coiled-coils are occasionally disordered, and this disordercan be critical to function, e.g. in the establishment ofmacromolecular assemblies (Gazi et al., 2008).

The T3SS apparatus and overview of the availablestructural information

The T3SS pathway recruits (Tampakaki et al., 2004): (i) aspecialized multi-component nanomachinery which isintegrated into the two bacterial membranes and includesthe main conductor channel and elements for substrateselection (Fig. 1E, red, orange, yellow parts), (ii) secretionsubstrates that assemble in the extracellular part (Fig. 1E,green, cyan, blue parts) or act in the extracellular space,(iii) chaperones required for efficient secretion (Fig. 1E,yellow ribbons), (iv) effectors interacting in the host cellcytoplasm with host cell proteins (Fig. 1E, magentaribbons) and (v) transcription regulators.

Recent studies have provided a wealth of structuralinformation: the structures of HrcQB-C (Fadouloglou et al.,2004), EscU (Zarivach et al., 2008) and EscN (Zarivachet al., 2007) provide insights into the properties of con-served core components. The structure of the central partof the C-ring component FliM is also known (Park et al.,2006). The inner membrane part of the Salmonella typhi-murium injectisome has been studied by electron micro-scopy (Marlovits et al., 2004; 2006). The crystal structureof EscJ, an inner membrane part component from the

Fig. 1. A–D. Antiparallel coiled-coils with assignment of heptad positions a–g. Positions normally occupied by hydrophobic/hydrophilic residuesare coloured yellow/red respectively. Positions with preferences for all types of residues are coloured blue. Canonical heptad repeats areshown for (A) dimers, (B) 3-a-helical bundles, (C) 4-a-helical bundles, (D) side-chain packing in the core of the ColE1 Rop (Banner et al.,1987) showing positions a and d which form flat slices, equally spaced along the bundle.E. Right: A schematic representation of a fully developed T3SS injectisome. Dark red: ATPase; orange: cytoplasmic ring (C-ring); yellow: innerand outer rings based on cryo-electron microscopy reconstruction; green: cryo-electron microscopy reconstruction of the S. flexneri needle(Deane et al., 2006); cyan: tip of the needle based on the LcrV molecular structure (PDB-1r6f); blue: translocator pore formed at the hostmembrane. Left: 3D structures of proteins/complexes with coiled-coil domains coloured according to their location/function. Green: needleproteins; cyan: needle tip proteins; purple: effectors; yellow: T3SS chaperones acting inside the bacterial cytoplasm (see text for details); grey:eukaryotic proteins targeted by effectors. Red segments indicate structural disorder as predicted by FoldIndex (window size of 50 residues).For the intrinsically disordered HrpO protein, the model is based on SAXS data.

720 A. D. Gazi, S. N. Charova, N. J. Panopoulos and M. Kokkinidis

© 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 719–729

Coiled-coil flexibility in type III secretion systems 721


enteropathogenic Escherichia coli (EPEC) is also avail-able (Yip et al., 2005a). The outer supramolecular struc-ture of the needle has been studied (Cordes et al., 2003),while structures of needle subunits from various bacteriahave been recently determined (Deane et al., 2006;Zhang et al., 2006; Wang et al., 2007).

At the tip of the T3SS needle resides an adaptor struc-ture which mediates the interaction between the needleand the translocation pore at the eukaryotic membrane.The adaptor is formed through polymerization of a singleprotein. Information is available for the following needle tipproteins from three T3SS families: IpaD (Shigella flex-neri), SipD (Salmonella spp.) and BipD (Burkholderiapseudomallei) from the Inv-Mxi-Spa family; LcrV (Yersiniaspp.), PcrV (Pseudomonas aeruginosa) and AcrV (Aero-monas salmonicida) from the Ysc family; EspA (EPEC)from the Ssa-Esc family. The structures of IpaD, BipD,LcrV and part of the EspA structure have been elucidated(Derewenda et al., 2004; Yip et al., 2005b; Espina et al.,2007; Johnson et al., 2007), while a 3D reconstruction ofthe MxiH filament is available (Deane et al., 2006).

Structures of 19 T3SS effectors from plant and animalpathogens are known (Stebbins, 2005; Desveaux et al.,2006), as well as 10 class I chaperone structures (Lilicet al., 2006), one class II chaperone (Buttner et al., 2008)and the class III chaperones–substrate complexes PscE/PscG/PscF (Quinaud et al., 2007) and YscE/YscG/YscF(Sun et al., 2008).

The occurrence of coiled-coil domains andstructural disorder in T3SS

Analyses of T3SS protein sequences (Table S1) reveal anunusually frequent occurrence of heptad repeats, which is

indicative for coiled-coil formation (Pallen et al., 1997;Delahay and Frankel, 2002). Recent structural work hasconfirmed the prevalence of coiled-coils (Fig. 1E) andcoiled-coil interactions in T3SS [e.g. in the Yersiniacomplex TyeA–YopN (Schubot et al., 2005) and in theHrpO and FliJ complexes with their cognate proteintargets (Gazi et al., 2008)]. T3SS proteins and coiled-coildomains are frequently predicted to be structurally disor-dered (supplemental Tables S1 and S2). For the 50 N-terminal residues of many T3SS effectors disorder is alsopredicted. These aspects will be elaborated in more detailbelow.

Cytoplasmic T3SS proteins

The most extensive heptad repeat pattern occurs in theHrpO/FliJ/YscO family of proteins (Gazi et al., 2008).Despite the absence of a significant homology, the familymembers share specific characteristics, e.g. coiled-coilpropensity and intrinsic disorder (Table S1, Fig. 2A). HrpOexhibits high a-helical content with coiled-coil character-istics and molten globule-like properties (Gazi et al.,2008). HrpO interacts, probably via coiled-coil formation,with HrpE, a highly a-helical T3SS protein. HrpE belongsto the FliH/YscL/HrpE/NolV family of T3SS proteins. Itsflagellar counterpart FliH is a regulator of the FliI ATPase(Minamino and Macnab, 2000; Minamino et al., 2002;Lane et al., 2005). Evidence from HrpO and its analoguesfrom all T3SS families and the flagellum suggests that theextreme flexibility (Fig. 2A) and propensity for coiled-coilinteractions of this class of intrinsically disordered pro-teins might be important elements in the establishment ofprotein–protein interaction networks required for T3SSfunction (Gazi et al., 2008).

Fig. 2. Sequence diagrams depicting predicted coiled-coil regions, transmembrane helices and structural disorder for (A) the HrpO/FliJ/YscOfamily of proteins and (B) the translocators from various T3SS families. Predicted disordered segments are coloured red, coiled-coil domainscyan, and transmembrane helices yellow. All sequences were predicted to be almost entirely a-helical (confirmed for HrpO by CD). Thesequences are drawn on a scale, reflecting their length. In A the sequences vary from 166 (HrpD) to 125 residues (SsaO), in B from 806(HrpF) to 363 residues (IpaC). Sequences were retrieved from NCBI (accession numbers: NP_395175 YscO; AAC25065 HrpO; CAD18022HrpD; NP_460381 SsaO; NP_461814 InvI; NP_220189 CT670; BAB52653 Y4yJ; P52613 FliJ; NP_863512 YopB; NP_858261 IpaB;AAP78990 IpaC; AAC38395 EspD; YP_001654911 CopB; YP_001654325 CopB2; AAF71482 HrpK; AAB86527 HrpF; P55711 NopX).



Chaperones and their complexes with export substrates

Many T3SS proteins have a pre-secretory requirement forsmall cytosolic chaperones.

Class I chaperones are small, dimeric proteins with atwo-layer sandwich fold and belong to the homologoussuperfamily 3.30.1460.10 according to the CATH proteinstructure classification (Orengo et al., 1997); dimerizationoccurs in a coiled-coil-like fashion via a central pair ofa-helices. They bind to T3SS effectors through addition ofa N-terminal b-strand of the effector to the chaperoneb-sheet (Lilic et al., 2006). The substrate is extensivelyunstructured. Class II chaperones bind translocation pro-teins. They posses tetratricopeptide repeats (TPR) whichform a curved layer of a-helices (Pallen et al., 2003;Buttner et al., 2008) in which coiled-coil-like interactionsand short, distorted helical bundles are established.

Class III chaperones are a heterogeneous group ofproteins that bind to subunits of the needle, the needle tipand the export substrates of the flagellum filament. Theyshare a common mode of substrate binding mediated bycoiled-coil interactions. For PscF and YscF, the majorneedle components of P. aeruginosa and Yersinia spp.,respectively, polymerization in the bacterial cytoplasm isprevented through binding to chaperones PscE/PscG andYscE/YscG respectively (Quinaud et al., 2007). In thePscG/PscE/PscF complex the C-terminal helix of PscF isstabilized in a helical bundle formed by part of the PscGTPR repeats through coiled-coil interactions (Quinaudet al., 2007).

Similarly, polymerization of the EPEC needle tip proteinEspA is prevented through binding with the class III chap-erone CesA. The CesA–EspA complex is a distorted 4-a-helical bundle (Yip et al., 2005b) with extensive coiled-coilinteractions.

T3SS needle proteins

The major extracellular T3SS component is the needlewith a length of 60 nm and a diameter of 7 nm for animalpathogens; a much longer structure (up to 2 mm) namedthe Hrp pilus is the needle counterpart in phytopathogenicbacteria (He and Jin, 2003; Cordes et al., 2003; Barrettet al., 2008). These structures are formed through thehelical assembly of multiple copies of a small a-helicalprotein. Along the needle axis runs a narrow (2.5 nm)conduit which is used for the passage of needle compo-nents, tip proteins, translocators and effectors, whereby apartially unfolded form of the substrate is required.

The Hrp pilus subunits are generally predicted to bealmost entirely a-helical, with the exception of thePseudomonas syringae species, for which the N-terminaldomain of the pilus subunit is predicted to containb-strands (Koebnik, 2001; He and Jin, 2003). No struc-

tural information is presently available for pilus-forming orneedle components of the Rhizobiales and ChlamydialesT3SS families.

Structures are available (Deane et al., 2006; Zhanget al., 2006; Wang et al., 2007) for three needle compo-nents from animal pathogens: MxiH (S. flexneri), BsaL(B. pseudomallei) and PrgI (S. typhimurium). These struc-tures are highly a-helical, with a central coiled-coil whichis essential for needle assembly. Outside this coiled-coil,all three proteins have highly mobile N-termini andC-termini, although these regions may retain somedegree of helical structure in solution (Blocker et al.,2008). The sequences of the coiled-coil parts of theneedle proteins show strong similarities, suggesting thatthey all share a common fold (Wang et al., 2007; Zhanget al., 2006). Circular dichroism (CD) spectra and thermalunfolding studies reveal that at temperatures (37°C) andconditions resembling the physiological ones, all threeC-terminally truncated proteins adopt a molten globule-like state; at higher temperatures their tertiary structurecollapses while the secondary structure is largely retained(Barrett et al., 2008). A molten globule-like/partiallyunfolded state of these proteins could be functionallyimportant, e.g. for transversing the needle channel and forthe extracellular assembly.

Analysis of structures and sequences of needle com-ponents from various pathogens suggests that the major-ity has a propensity for structural disorder (Table S2).

The needle appears to play a major role in host sensingand signal transmission from the distal to the basal end ofT3SS (Deane et al., 2006). Signal transmission is sus-pected to utilize the structural flexibility of the needlesubunits (Kenjale et al., 2005; Torruellas et al., 2005;Deane et al., 2006; Barrett et al., 2008).

Needle tip proteins

The structures of LcrV, IpaD and BipD exhibit a commontopology with a central, long coiled-coil formed betweenthe C-terminal helix and a helix in the preceding part of thesequence. An additional domain is inserted betweenthese two helices, and frequently a further domain isformed at the extreme N-terminus (Derewenda et al.,2004; Erskine et al., 2006; Johnson et al., 2007; Blockeret al., 2008). Parts of the central a-helices occasionallyform coiled-coil interactions with other helices of the struc-ture, giving rise to local three- or four a-helical bundles. Aninherent flexibility, correlated with chaperoning, has beenobserved in coiled-coil regions involved in subunit poly-merization. In LcrV flexibility manifests itself as a kink in itsC terminal helix which resembles the hinge identified inone form of the S. flexneri needle component MxiH (Dere-wenda et al., 2004; Yip et al., 2005b; Deane et al., 2006).It has been suggested that this flexibility could be relevant



to tip assembly or transmission of signals to the rest ofT3SS (Blocker et al., 2008). Analysis of the tip proteinsequences is consistent with the experimental observa-tions as it predicts a propensity for structural disorder intheir coiled-coil regions (Table S2).

The complex of the tip protein EspA with its chaperoneCesA is stabilized by coiled-coil interactions which giverise to a 4-a-helical bundle (Yip et al., 2005b). The tipassembled by EspA is a long filament (Daniell et al.,2001); it has been suggested that the coiled-coil regionsof EspA are the basis for filament assembly (Yip et al.,2005b). For the tip proteins of the Inv-Mxi-Spa T3SSfamily, the N-terminal part acts as a chaperone for thecoiled-coil domain preventing oligomerization in the bac-terial cytoplasm (Johnson et al., 2007). For LcrV the chap-eroning function is associated with LcrG which binds oneof the LcrV helices of the central coiled-coil structure(Lawton et al., 2002; Hamad and Nilles, 2007).

For tip proteins of the Inv-Mxi-Spa family it has beensuggested that after exit from the needle in a partiallyunfolded form, the N-terminus is repositioned exposingthe coiled-coil and promoting the oligomerization of thecomplex while the C-terminal part interacts with theneedle (Derewenda et al., 2004; Nilles, 2004; Johnsonet al., 2007).

In summary, tip proteins are highly interactive with apronounced propensity for structural flexibility/intrinsicdisorder.

Translocators

The translocator pore is formed in the plasma membraneof the eukaryotic target cell (Tampakaki et al., 2004) andis essential for the delivery of effectors directly into the cellcytoplasm. Its components (translocators) are usually twoproteins in animal pathogens (e.g. YopB and YopD inYersinia spp.), while in most plant pathogens only onetranslocator protein is found (e.g. HrpK in P. syringae).

Despite low sequence similarity and variable sequencelengths, the translocators are predicted to possess coiled-coil domains, transmembrane helices (Buttner andBonas, 2002; Delahay and Frankel, 2002) and a profoundpropensity for a-helix formation (Fig. 2B). Fold recognitionidentifies consistently long coiled-coil domains (> 75 resi-dues) as templates for all translocators from animal patho-gens (Table S3), the best template being the methyl-accepting chemotaxis protein domain (Park et al., 2006),a dimeric 4-a-helical bundle [Protein Data Bank (PDB) id:2ch7]. FarUV CD spectra from animal pathogenic trans-locators are typical for coiled-coil (Lees et al., 2006).Translocators from plant pathogens exhibit a reduced pro-pensity for long coiled-coils. A pronounced propensity forstructural disorder (Fig. 2B) is predicted for translocatorsand is confirmed by NearUV CD spectra (Hamada et al.,

2005). The intrinsic flexibility of these molecules may be aprerequisite for their function which includes secretion,subsequent insertion to the host cell membrane and occa-sionally effector activity (Hayward and Koronakis, 1999).

T3SS effectors

Of the 19 effector structures determined so far, six formregular coiled-coils, while several others exhibit shortheptad repeat patterns in their sequences which give riseto coiled-coil interactions and short, distorted a-helicalbundles: the YpkA subdomain comprising residues 434–615 folds in two 3-helical bundles (Prehna et al., 2006).Coiled-coils are also observed for the N-terminal domainsof YopH and SptP (Stebbins and Galan, 2000; Khandelwalet al., 2002) and more so for MxiC and YopN-TyeA(Schubot et al., 2005; Deane et al., 2008). A non-typicalantiparallel 3-a-helical bundle is adopted by AvrPto (Wulfet al., 2004) which shows a considerable plasticity invarious structural studies (PDB ids: 1R5E, 2QKW); thisis consistent with the disorder predicted by FoldIndex.Although interaction with the target protein Pto kinaseoccurs via b-strand addition (Xing et al., 2007), thea-helices of AvrPto may be used in interactions with othercoiled-coils of the host.

Regardless of the presence of coiled-coils, the mostcommon feature among effector structures is the disorderof their extreme N-terminal part where the secretion signalresides. Usually, the 15–20 N-terminal residues of effec-tors are disordered. Truncation of N- and C-terminal resi-dues was necessary for the NMR study of the AvrPtoeffector (Wulf et al., 2004), while in the crystal structure ofthe AvrPto–Pto kinase complex 28 N-terminal residuesare missing (Xing et al., 2007). Although, the full lengthAvrB and AvrPphF ORF2 were crystallized, electrondensity was not observed for the 27 N-residues in eachcase, probably due to disorder (Lee et al., 2004; Singeret al., 2004). Residues 1–22 and 45–51of the SipA effec-tor in the complex with the class I chaperone InvB arehighly disordered (Lilic et al., 2006). A large majority(~75%) of P. syringae pv tomato effectors show a signifi-cant propensity for structural disorder in the region of their50 N-terminal residues based on the ratio of order- versusdisorder-promoting residues (Table S1). A value of 0.58 isthe average ratio in proteomes based on the amino acidfrequencies of order- and disorder-promoting residues(Wagner et al., 2003); lower values indicate a propensityfor disorder. The only structured N-terminal region of aT3SS effector is that of YopH. The 129 residue N-terminaldomain has two functions: the first 70 residues contain theCBD domain for chaperone SycH, while the full 129N-terminal part binds to phosphotyrosine-containing pro-teins and adopts an overall globular fold (Khandelwalet al., 2002).



Comparison with other Gram-negative secretorysystems and conclusions

To compare with T3SS, coiled-coil predictions were alsocarried out for other secretion systems of selected bacte-ria, i.e. Helicobacter pylori (T4SS), Legionella pneumo-phila (T2SS and T4SS) and the type 4 pili (T4P) ofL. pneumophila and P. aeruginosa.

For 2878 proteins encoded in the L. pneumophilagenome (GenBank accession number NC_006369), thepredicted coiled-coil content is 9%. Using the VirulenceFactor Database (Yang et al., 2008) classification ofL. pneumophila proteins, a coiled-coil content of 13%is predicted for T2SS (11 proteins), 8% for T4ASS (11proteins), 8% for T4BSS (25 proteins), 11% for T4P(10 proteins) and strikingly 24% for T4BSS effectors(38 proteins).

For 1536 proteins encoded in the H. pylori genome(NC_008086) a coiled-coil content of 15% is predicted,with 25% for T4ASS (29 proteins) and 35% for the knownT4ASS effector (1 protein). By comparison the H. pyloriflagellum (40 proteins) has a predicted content of 22%.

For 5566 proteins encoded in the P. aeruginosagenome (NC_002516) the predicted content is 7%, 10%for T4P and for comparison 20% for T3SS and 17% for theflagellum.

In can be thus summarized from this preliminary analy-sis that relatively high coiled-coil content is predicted forsome other secretory systems, and in particular for theT4ASS of H. pylori and for the L. pneumophila T4BSSeffectors. The latter exhibit also a particularly high propen-sity for structural disorder (on the average 42% disor-dered regions according to the FOLDINDEX analysis), aproperty they share with the T3SS effectors.

Discrepancies in the predicted T4ASS coiled-coilcontent between L. pneumophila and H. pylori could pos-sibly reflect differences in the annotation of the twogenomes. From the available structures of T4SS proteins,only the E. coli Type IV pilus tip protein (PDB code 1R8I)has significant coiled-coil content and considerable disor-der (Fig. 1E). In the T4P structure, the centre of the fibreconsists of closely packed coiled-coil layers assembled bypilin subunits (Forest and Tainer, 1997).

In conclusion, recent structural studies, combined withthe in silico analysis of bacterial genomes, have consid-erably consolidated our understanding of the structuraland functional versatility of coiled-coil domains in T3SSand are beginning to do so also for other secretorysystems. The occurrence (either derived from proteinstructures or predicted) of coiled-coils in T3SS is consid-erably higher than their average predicted occurrence inprokaryotic proteomes (Schubot et al., 2005). They occurin all types of T3SS proteins, including effectors of plantpathogens, for which in earlier studies no coiled-coils

could be predicted (Delahay and Frankel, 2002). The3D-structures of T3SS proteins show that commonly usedpredictive methods frequently tend to underestimate theoccurrence of coiled-coils, e.g. in the case of the effectorprotein AvrPto1 (Table S1). Other examples include theneedle protein Bsal, the chaperones SycD and YscG, andthe tip protein LcrV (Table S2). A widespread feature ofcoiled-coil-containing T3SS proteins is a considerablestructural flexibility and disorder, a property which can befrequently predicted from the amino acid compositionof their sequences. Disorder manifests itself either asmissing parts of crystallographically determined struc-tures due to weak electron density, as it is the case withthe N-termini of effectors, or occasionally as a moltenglobule-like state at conditions resembling the physiologi-cal ones, e.g. as in the case of HrpO. Furthermore,structural flexibility frequently gives rise to a significantmalleability of coiled-coil domains which becomes evidentin the case of multiple structural studies of the sameprotein. For example, AvrPto (PDB ids: 2QKW, 1R5E)exhibits a remarkable plasticity of its core, due to rear-rangements of the a-helical bundle. Repacking of hydro-phobic core residues of coiled-coil domains has beenoccasionally reported for other systems, e.g. in the caseof the RNA-binding ColE1Rop protein and its mutants(Glykos et al., 1999; 2006).

Coiled-coiled propensity and intrinsic structural disorderare frequently essential prerequisites for the establish-ment of functional interaction networks in T3SS asexemplified by the HrpO protein and its flagellarcounterpart FliJ. In addition, the assembly of the T3SSsupramolecular structures, e.g. the needle, may occurthrough the stepwise polymerization of a major subunit(e.g. MxiH, BsaL and PrgI) via a flexible or partially dis-ordered C-terminal helix with a propensity for coiled-coilinteractions.

The N-terminal sequences of T3SS effectors exhibitfrequently specific biases in their order- and disorder-promoting residues, resulting in a disorder propensity.Strikingly, these disorder-associated biases result inN-terminal sequence preferences which are similar tothose determined in earlier studies for effectors fromvarious bacterial species (Greenberg and Vinatzer, 2003).The structural disorder of the N-termini may thus play arole as a secretion signal.

Earlier studies (Akeda and Galan, 2005) which suggesta central role for T3SS ATPases in protein secretion (sub-strate recognition, chaperone release and unfolding ofT3SS substrates) support this hypothesis, as they implythat N-terminal disorder facilitates recognition and unfold-ing of secretion substrates. As structural disorder does notensure specificity of substrate recognition, it may beassumed that N-terminal flexibility could be one ofmultiple secretion signals (Marlovits et al., 2006), with



other signals, e.g. chaperones, ensuring specificity. Flex-ible or disordered coiled-coil domains could facilitate rapidunfolding before secretion. As shown in the case of theHrpO protein, proteins exhibiting coiled-coil propensityare capable of adopting highly non-globular/unfolded con-formations, while maintaining a considerable a-helicalcontent. The geometrical dimensions of such non-globularhelical conformations permit passage through the narrowneedle channel which serves as a conduit for secretion,provided that the appropriate secretion signal is present. Itis intuitive to assume that after passing this conduit, suchpre-formed and folding-competent helices encompassinga few turns may form a nucleation site which promotesassembly of a globular coiled-coil domain. Flexible coiled-coil domains are thus particularly suitable as secretionsubstrates as they can easily unfold into secretion-competent a-helices, which in turn may refold into a nativestructure following a relatively simple pathway. Thisconcept, along with the results of comprehensive func-tional studies outlined in this review, may offer an expla-nation for the prevalence of coiled-coils in T3SS. Thepredicted high occurrence of coiled-coil domains andstructural disorder in other secretion systems, and in par-ticular in T4BSS effectors might indicate that this concepthas also some validity in other Gram-negative secretorymechanisms.

Experimental procedures

Sequence analysis

Protein sequences retrieved from the NCBI/GenBank andfrom the PPI: P. syringae Genome Resources (http://www.pseudomonas-syringae.org) were analysed for intrinsic disorder[program FoldIndex© (Prilusky et al., 2005)], coiled-coil propensi-ties [COILS (Lupas et al., 1991) and MATCHER (Fischetti et al.,1993)] and secondary structure [PSIPRED (Jones, 1999)].Fold recognition and sequence threading analysis usedPHYRE (Kelley et al., 2000; Bennett-Lovsey et al., 2008) andmGenTHREADER (McGuffin and Jones, 2003). Predictions fortransmembrane a-helices used MEMSAT3 (Jones, 2007). Thepropensity of N-termini for disorder was analysed on the basis oftheir content of order-/disorder-promoting residues (Dunker et al.,2002). 3-D structures of proteins were retrieved from the PDB.Helical packing was analysed with SOCKET (Walshaw andWooifson, 2001). Molecular graphics were produced with PyMOL(DeLano Scientific). The Virulence Factor Database (Yang et al.,2008) was used to classify proteins from selected bacterial patho-gens to Gram-negative secretory systems (II–IV) and functions(e.g. effectors).

Acknowledgements

This work was supported by PENED, PYTHAGORAS and PEP(KP-15) grants from the Greek Ministry of Education, GSRT andthe EPEAEK-Plant Molecular Biology and Biotechnology and theProtein Biotechnology graduate programs. S.N.C. was recipientof an Onassis Foundation fellowship.

References

Abramovitch, R.B., Anderson, J.C., and Martin, G.B. (2006)Bacterial elicitation and evasion of plant innate immunity.Nature Rev Mol Cell Biol 7: 601–610.

Akeda, Y., and Galan, J.E. (2005) Chaperone release andunfolding of substrates in type III secretion. Nature 437:911–915.

Alfano, J.R., and Collmer, A. (2004) Type III secretion systemeffector proteins: double agents in bacterial disease andplant defense. Annu Rev Phytopathol 42: 385–414.

Banner, D.W., Kokkinidis, M., and Tsernoglou, D. (1987)Structure of the ColE1 Rop protein at 1.7 Å resolution.J Mol Biol 196: 657–675.

Barrett, B.S., Picking, W.L., Picking, W.D., and Middaugh,C.R. (2008) The response of type three secretion systemneedle proteins MxiH (Delta5), BsaL (Delta5), and PrgI(Delta5) to temperature and pH. Proteins 73: 632–643.

Bennett-Lovsey, R.M., Hebert, A.D., Sternberg, M.J.E., andKelley, L.A. (2008) Exploring the extremes of sequence/structure space with ensemble fold recognition in theprogram Phyre. Prot Struct Funct Bioinf 70: 611–625.

Blocker, A.J., Deane, J.E., Veenendaal, A.K.J., Roversi, P.,Hodgkinson, J.L., Johnson, S., and Lea, S.M. (2008)What’s the point of the type III secretion system needle?Proc Natl Acad Sci USA 105: 6507–6513.

Brown, P.N., Mathews, M.A.A., Joss, L.A., Hill, C.P., andBlair, D.F. (2005) Crystal structure of the flagellar rotorprotein FliN from Thermotoga maritima. J Bacteriol 187:2890–2902.

Buttner, D., and Bonas, U. (2002) Port of entry – the type IIIsecretion translocon. Trends Microbiol 10: 186–192.

Buttner, C.R., Sorg, I., Cornelis, G.R., Heinz, D.W., andNiemann, H.H. (2008) Structure of the Yersinia entero-colitica type III secretion translocator chaperone SycD.J Mol Biol 375: 997–1012.

Cascales, E., and Christie, P.J. (2003) The versatile bacterialtype IV secretion systems. Nat Rev Microbiol 1: 137–149.

Cordes, F.S., Komoriya, K., Larquet, E., Yang, S., Egelman,E.H., Blocker, A., and Lea, S.M. (2003) Helical structure ofthe needle of the type III secretion system of Shigellaflexneri. J Biol Chem 278: 17103–17107.

Daniell, S.J., Takahashi, N., Wilson, R., Friedberg, D., Rosen-shine, I., Booy, F.P., et al. (2001) The filamentous type IIIsecretion translocon of enteropathogenic Escherichia coli.Cell Microbiol 3: 865–871.

Deane, J.E., Roversi, P., Cordes, F.S., Johnson, S., Kenjale,R., Daniell, S., et al. (2006) Molecular model of a type IIIsecretion system needle: Implications for host-cell sensing.Proc Natl Acad Sci USA 103: 12529–12533.

Deane, J.E., Roversi, P., King, C., Johnson, S., and Lea,S.M. (2008) Structures of the Shigella flexneri Type 3secretion system protein Mxic reveal conformational vari-ability amongst homologues. J Mol Biol 377: 985–992.

Delahay, R.M., and Frankel, G. (2002) Coiled-coil proteinsassociated with type III secretion systems: a versatiledomain revisited. Mol Microbiol 45: 905–916.

Derewenda, U., Mateja, A., Devedjiev, Y., Routzahn, K.M.,Evdokimov, A.G., Derewenda, Z.S., and Waugh, D.S.(2004) The structure of Yersinia pestis V-antigen, anessential virulence factor and mediator of immunity againstplague. Structure 12: 301–306.



http://www.pseudomonas-syringae.org


Desveaux, D., Singer, A.U., and Dangl, J.L. (2006) Type IIIeffector proteins: doppelgangers of bacterial virulence.Current Opinion Plant Biology 9: 376–382.

Ding, Z., Atmakuri, K., and Christie, P.J. (2003) The outs andins of bacterial type IV secretion substrates. Trends Micro-biol 11: 527–535.

Dunker, A.K., Brown, C.J., Lawson, J.D., Iakoucheva, L.M.,and Obradovic, Z. (2002) Intrinsic disorder and proteinfunction. Prot Func Biochem 41: 6573–6582.

Dyson, H.J., and Wright, P.E. (2005) Intrinsically unstructuredproteins and their functions. Nat Rev Mol Cell Biol 6: 197–208.

Erskine, P.T., Knight, M.J., Ruaux, A., Mikolajek, H., Sang,N.W.F., Withers, J., et al. (2006) High resolution structureof BipD: an invasion protein associated with the type IIIsecretion system if Burkholderia pseudomallei. J Mol Biol363: 125–136.

Espina, M., Ausar, F., Middaugh, C.R., Baxter, M.A., Picking,W.D., and Picking, W.L. (2007) Conformational stability anddifferential structural analysis of LcrV, PcrV, BipD and SipDfrom type III secretion systems. Protein Sci 16: 704–714.

Fadouloglou, V.E., Tampakaki, A.P., Glykos, N.M., Bastaki,M.N., Hadden, J.M., Phillips, S.E., et al. (2004) Structure ofHrcQB-C, a conserved component of the bacterial type IIIsecretion systems. Proc Natl Acad Sci USA 101: 70–75.

Fischetti, V.A., Landau, G.M., Schmidt, J.P., and Sellers, P.(1993) Identifying periodic occurrences of a template withapplications to protein structure. Inform Process Lett 45:11–18.

Forest, K.T., and Tainer, J.A. (1997) Type-4 pilus-structure:outside to inside and top to bottom – a minireview. Gene192: 165–169.

Gazi, A.D., Bastaki, M., Charova, S.N., Gkougkoulia, E.A.,Kapellios, E.A., Panopoulos, N.J., and Kokkinidis, M.(2008) Evidence for a widespread interaction mode of dis-ordered proteins in bacterial type III secretion systems.J Biol Chem 283: 34062–34068.

Glykos, N.M., Cesareni, G., and Kokkinidis, M. (1999) Proteinplasticity to the extreme: changing the topology of a4-a-helical bundle with a single amino acid substitution.Structure 7: 597–603.

Glykos, N.M., Papanikolau, Y., Vlassi, M., Kotsifaki, D.,Cesareni, G., and Kokkinidis, M. (2006) Loopless Rop:structure and dynamics of an engineered homotetramericvariant of the repressor of primer protein. Biochemistry 45:10905–10919.

Greenberg, J.T., and Vinatzer, B.A. (2003) Identifying type IIIefectors of plant pathogens and analysing their interactionwith plant cell. Curr Opin Microbiol 6: 20–28.

Grigoryan, G., and Keating, A.E. (2008) Structural specificityin coiled-coil interactions. Curr Opin Struct Biol 18: 1–7.

Hamad, M.A., and Nilles, M.L. (2007) Structure-functionanalysis of the c-terminal domain of LcrV from Yersiniapestis. J Bacteriol 189: 6734–6739.

Hamada, D., Kato, T., Ikegami, T., Suzuki, K.N., Hayashi, M.,Murooka, Y., et al. (2005) EspB from enterohaemorrhagicEscherichia coli is a natively partially folded protein. FEBSJ 272: 756–768.

Hayward, R.D., and Koronakis, V. (1999) Direct nucleationand bundling of actin by the SipC protein of invasive Sal-monella. EMBO J 18: 4926–4934.

He, S.Y., and Jin, Q. (2003) The Hrp pilus: learning fromflagella. Curr Opin Microbiol 6: 15–19.

Hueck, C.J. (1998) Type III protein secretion systems in bac-terial pathogens of animals and plants. Microbiol Mol BiolRev 62: 379–433.

Imada, K., Minamino, T., Tahara, A., and Namba, K. (2007)Structural similarity between the flagellar type III ATPaseFliI and F1-ATPase subunits. Proc Natl Acad Sci USA 104:485–490.

Johnson, S., Roversi, P., Espina, M., Olive, A., Deane, J.E.,Birket, S., et al. (2007) Self-chaperoning of the type IIIsecretion system needle tip proteins IpaD and BipD. J BiolChem 282: 4035–4044.

Jones, D.T. (1999) Protein secondary structure predictionbased on position-specific scoring matrices. J Mol Biol 292:195–202.

Jones, D.T. (2007) Improving the accuracy of transmem-brane protein topology prediction using evolutionary infor-mation. Bioinf 23: 538–544.

Kelley, L.A., MacCallum, R.M., and Sternberg, M.J.E. (2000)Enhanced genome annotation with structural profiles in theprogram 3D-PSSM. J Mol Biol 299: 499–500.

Kenjale, R., Wilson, J., Zenk, S., Saurya, S., Picking, W.L.,Picking, W.D., and Blocker, A. (2005) The needle compo-nent of the type III secretion of Shigella regulates theactivity of the secretion apparatus. J Biol Chem 280:42929–42937.

Khandelwal, P., Kellikuli, K., Smith, C.L., Saper, M.A., andZuiderweg, E.R.P. (2002) Solution structure and phop-sphopeptide binding to the N-terminal domain of YersiniaYopH: comparison with a crystal structure. Biochemistry41: 11425–11437.

Koebnik, R. (2001) The role of bacterial pili in protein andDNA translocation. Trends Microbiol 9: 586–590.

Lane, M.C., O’Toole, P.W., and Moore, S.A. (2005) MolecularBasis of the Interaction between the Flagellar Export Pro-teins FliI and FliH from Helicobacter pylori. J Biol Chem281: 508–517.

Lawton, D.G., Longstaff, C., Wallace, B.A., Hill, J., Leary,S.E.C., Titball, R.W., and Brown, K.A. (2002) Interactions ifthe type III secretion pathway proteins LcrV and LcrG fromYersinia pestis are mediated by coiled-coil domains. J BiolChem 277: 38714–38722.

Lee, C.C., Wood, M.D., Ng, K., Luginbuhl, P., Spraggon, G.,and Katagiri, F. (2004) Crystal structure of the Type IIIeffector AvrB from Pseudomonas syringae. Structure 12:487–494.

Lees, J.G., Miles, A.J., Wien, F., and Wallace, B.A. (2006) Areference database for circular dichroism spectroscopycovering fold and secondary structure space. Bioinformat-ics 22: 1955–1962.

Lilic, M., Vujanac, M., and Stebbins, C.E. (2006) A commonstructural motif in the binding of virulence factors to bacte-rial secretion chaperones. Mol Cell 21: 653–664.

Liu, J., and Rost, B. (2001) Comparing structure and functionbetween entire genomes. Prot Sci 10: 1970–1979.

Lupas, A. (1996) Coiled coils: new structures and new func-tions. Trends Biochem Sci 21: 375–382.

Lupas, A., Van Dyke, M., and Stock, J. (1991) Predictingcoiled coils from protein sequences. Science 252: 1162–1164.



McGuffin, L.J., and Jones, D.T. (2003) Improvement of theGenTHREADER method for genomic fold recognition.Bioinformatics 19: 874–881.

Marlovits, T.C., Kubori, T., Sukkan, A., Thomas, D., Galan,J.E., and Unger, V.M. (2004) Structural insights into theassembly of the type III secretion needle complex. Science5: 1040–1042.

Marlovits, T.C., Kubori, T., Lara-Tejero, M., Thomas, D.,Unger, V.M., and Galan, J.E. (2006) Assembly of the innerrod determines needle length in the type III secretion injec-tisome. Nature 441: 637–640.

Minamino, T., and Macnab, R.M. (2000) FliH, a soluble com-ponent of the type III flagellar export apparatus of Salmo-nella, forms a complex with FliI and inhibits its ATPaseactivity. Mol Microbiol 37: 1494–1503.

Minamino, T., Gonzalez-Pedrajo, B., Oosawa, K., Namba, K.,and Macnab, R.M. (2002) Structural properties of FliH, anATPase regulatory component of the Salmonella type IIIflagellar export apparatus. J Mol Biol 322: 281–290.

Namba, K. (2001) Roles of partly unfolded conformations inmacromolecular self-assembly. Genes Cells 6: 1–12.

Nilles, M.L. (2004) Dissecting the structure of LcrV from Yers-inia pestis, a truly unique virulence protein. Structure 12:357–358.

Odgren, P.R., Harvie, L.W., and Fey, E.G. (1996) Phyloge-netic occurrence of coiled coil proteins: implications fortissue structure in metazoa via a coiled coil tissue matrix.Proteins 24: 467–484.

Orengo, C.A., Michie, A.D., Jones, D.T., Swindells, M.B., andThornton, J.M. (1997) CATH: a hierarchic classification ofprotein domain structures. Structure 5: 1093–1108.

Paliakasis, C.D., and Kokkinidis, M. (1992) Relationshipsbetween sequence and structure for the four-alpha-helixbundle tertiary motif in proteins. Protein Eng 5: 739–749.

Pallen, M.J., Dougan, G., and Frankel, G. (1997) Coiled-coildomains in proteins secreted by type III secretion systems.Mol Microbiol 25: 423–425.

Pallen, M.J., Francis, M.S., and Futterer, K. (2003)Tetratricopeptide-like repeats in type III secretion chaper-ones and regulators. FEMS Microbiol Lett 223: 53–60.

Pallen, M.J., Beatson, S.A., and Bailey, C.M. (2005) Bioin-formatics, genomics and evolution of non-flagellar type-IIIsecretion systems: a Darwinian perspective. FEMS Micro-biol Rev 29: 201–229.

Park, S., Lowder, B., Bilwes, A.M., Blair, D.F., and Crane,B.R. (2006) Structure of FliM provides insight into assem-bly of the switch complex in the bacterial flagella motor.Proc Natl Acad Sci USA 103: 11886–11891.

Prehna, G., Ivanov, M.I., Bliska, J.B., and Stebbins, C.E.(2006) Yersinia virulence depends on mimicry of host rho-family nucleotide dissociation inhibitors. Cell 126: 869–880.

Prilusky, J., Felder, C.E., Zeev-Ben Mordehai, T., Rydberg,E.H., Man, O., Beckmann, J.S., et al. (2005) FoldIndex©: asimple tool to predict whether a given protein sequence isintrinsically unfolded. Bioinformatics 21: 3435–3438.

Pugsley, A.P., Francetic, O., Possot, O.M., Sauvonnet, N.,and Hardie, K.R. (1997) Recent progress and future direc-tions in studies of the main terminal branch of the generalsecretory pathway in Gram-negative bacteria-a review.Gene 192: 13–19.

Quinaud, M., Ple, S., Job, V., Contreras-Martel, C., Simorre,J.P., Attree, I., and Dessen, A. (2007) Structure of theheterotrimeric complex that regulates Type III secretionneedle formation. Proc Natl Acad Sci USA 104: 7803–7808.

Schubot, F.D., Jackson, M.W., Penrose, K.J., Cherry, S.,Tropea, J.E., Plano, G.V., and Waugh, D.S. (2005) Three-dimensional structure of a macromolecular assembly thatregulates type III secretion in Yersinia pestis. J Mol Biol346: 1147–1161.

Singer, A.U., Desveaux, D., Betts, L., Chang, J.H., Nimchuk,Z., Grant, S.R. et al. (2004) Crystal structures of the TypeIII effector protein AvrPphF and its chaperone reveal resi-dues required for plant pathogenesis. Structure 12: 1669–1681.

Stebbins, C.E. (2005) Structural microbiology at thepathogen–host interface. Cell Microbiol 7: 1227–1236.

Stebbins, C.E., and Galan, J.E. (2000) Modulation of hostsignaling by a bacterial mimic: structure of the Salmonellaeffector SptP bound to Rac1. Mol Cell 6: 1449–1460.

Sugase, K., Dyson, H.J., and Wright, P.E. (2007) Mechanismof coupled folding and binding of an intrinsically disorderedprotein. Nature 447: 1021–1025.

Sun, P., Austin, B.P., Tropea, J.E., and Waugh, D.S. (2008)Structural characterization of the Yersinia pestis type IIIsecretion system needle protein YscF in complex with itsheterodimeric chaperone YscE/YscG. J Mol Biol 377: 819–830.

Tampakaki, A.P., Fadouloglou, V.E., Gazi, A.D., Panopoulos,N.J., and Kokkinidis, M. (2004) Conserved features of typeIII secretion. Cell Microbiol 6: 805–816.

Torruellas, J., Jackson, M.W., Pennock, J.W., and Plano,G.V. (2005) The Yersinia pestis type III secretion needleplays a role in the regulation of Yop secretion. Mol Microbiol57: 1719–1733.

Troisfontaines, P., and Cornelis, G.R. (2005) Type III secre-tion: more systems than you think. Physiology 20: 326–339.

Wagner, V.E., Bushnell, D., Passador, L., Brooks, A.I., andIglewski, B.H. (2003) Microarray analysis of Pseudomonasaeruginosa quorum-sensing regulons: effects of growthphase and environment. J Bacteriol 185: 2080–2095.

Walshaw, J., and Wooifson, D.N. (2001) Socket: a programfor identifying and analysing coiled-coil motifs withinprotein structures. J Mol Biol 307: 1427–1450.

Wang, Y., Ouellette, A.N., Egan, C.W., Rathinavelan, T., Im,W., and De Guzman, R.N. (2007) Differences in the elec-trostatic surfaces of the Type III secretion needle proteinsPrgI, BsaL, and MxiH. J Mol Biol 371: v.

Wulf, J., Pascuzzi, P.E., Martin, G.B., and Nicholson, L.K.(2004) The solution structure of type III effector proteinAvrPto reveals conformational and dynamic featuresimportant for plant pathogenesis. Structure 12: 1257–1268.

Xing, W.M., Zou, Y., Liu, Q., Hao, Q., Zhou, J.M., and Chai,J.J. (2007) The structural basis for activation of plant immu-nity by bacterial effector protein AvrPto. Nature 449: 243–247.

Yang, J., Chen, L.H., and Sun, L.L., Yu, J., and Jin, Q. (2008)VFDB 2008 release: an enchanced web-based resourcefor comparative pathogenomics. Nucleic Acids Res 36:D539–D542.



Yip, C.K., Kimbrough, T.G., Felise, H.B., Vuckovic, M.,Thomas, N.A., Pfuetzner, R.A., et al. (2005a) Structuralcharacterization of the molecular platform for type III secre-tion system assembly. Nature 435: 702–707.

Yip, C.K., Finlay, B.B., and Strynadka, N.C.J. (2005b) Struc-tural characterization of a type III secretion system filamentprotein in complex with its chaperone. Nat Struct Mol Biol12: 75–81.

Zarivach, R., Vuckovic, M., Deng, W., Finlay, B.B., andStrynadka, N.C.J. (2007) Structural analysis of a prototypi-cal ATPase from the type III secretion system. Nat StructMol Biol 14: 131–137.

Zarivach, R., Deng, W., Vuckovic, M., Felise, H.B., Nguyen,H.V., Miller, S.I., et al. (2008) Structural analysis of theessential self-cleaving type III secretion proteins EscU andSpaS. Nature 453: 124–128.

Zhang, L., Wang, Y., Picking, W.L., Picking, W.D., and DeGuzman, R.N. (2006) Solution structure of monomericBsaL, the Type III secretion needle protein of Burkholderiapseudomallei. J Mol Biol 359: 322–330.

Supporting information

Additional Supporting Information may be found in the onlineversion of this article:

Table S1. Disorder analysis and heptad repeat predictions forproteins from the T3SS of P. syringae pv tomato DC3000. For theheptad repeat prediction the MATCHER program was used. Forthe AvrPto1 protein the coiled-coil content derived from 3D struc-tures (PDB id: 2QKW and 1R5E) is given in parentheses. Theoverall protein disorder was calculated using the FOLDINDEX

program with a window of 21 residues. The N-terminal proteindisorder calculations were based on the Dunker et al. (2002)definition for order- and disorder-promoting residues in a givensequence. The available structural information on HrcQb doesnot include the disordered N-terminal domain. Effector andpilus/harpin protein sequences were retrieved from http://www.pseudomonas-syringae.org. Accession numbers for therest of the proteins examined are: HrpF: NP_791214, HrpJ:NP_791230, HrpD: NP_791212, HrpB: NP_791210, HrpP:NP_791225, HrpO: NP_791226, HrpG: NP_791215, HrpV:NP_791218, HrpE: NP_791213, HrcN: NP_791227, HrcQb:NP_791223.Table S2. Disorder analysis for T3SS proteins of known3D-structure with coiled-coil structure exceeding 30%. Theoverall protein disorder was calculated using the FoldIndexprogram with a window of 21 residues. The N-terminal proteindisorder calculations were based on the Dunker et al. (2002)definition for order- and disorder-promoting residues. UniProtaccession numbers for the proteins examined are: MxiH:P0A223, PrgI: P41784, BsaL: Q63K18, SycD: O87496, PscE:Q9I317, PscG: P95435, YscE: Q7ARI1, YscG: Q7ARH9, CesA:O52124, LcrV: P0C7U7, IpaD: P18013, BipD: Q63K37, EspA:Q47184.Table S3. Threading of various translocator sequences usingmGenTHREADER (McGuffin and Jones, 2003). The structure ofthe methyl-accepting chemotaxis protein domain (2ch7) is fre-quently selected as template for translocators from animal-associated bacteria.

Please note: Wiley-Blackwell are not responsible for the contentor functionality of any supporting materials supplied by theauthors. Any queries (other than missing material) should bedirected to the corresponding author for the article.





Coiled-coils in type III secretion systems: structural flexibility, disorder and biological...

Documents

Transcript of Coiled-coils in type III secretion systems: structural flexibility, disorder and biological...