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The mechanism of pore formation by bacterial toxins
The mechanism of pore formation by bacterial toxins Sarah J Tilley and Helen R Saibil A remarkable group of proteins challenge the notions that protein sequence determines a unique three-dimensional structure, and that membrane and soluble proteins are very distinct. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. Recent structural studies provide ideas about how these changes take place. The recently solved structures of the b-pore-forming toxins LukS, e-toxin and intermedilysin confirm that the pore-forming regions are initially folded up on the surfaces of the soluble precursors. To create the transmembrane pores, these regions must extend and refold into membrane-inserted b-barrels. Addresses School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK Corresponding author: Saibil, Helen R ([email protected])
members of this family are still very unclear. The bacterial toxin colicin targets Escherichia coli cells by making pores that leak ions, depolarizing the membrane and ultimately causing cell death. Colicins usually contain three domains that are involved in receptor binding, transport and killing. Depending on the colicin, the toxic (killing) domain either inhibits protein or peptidoglycan synthesis, or has nuclease or pore-forming activity. The other domains are used to bind and translocate the toxic domain into the bacterial cell. Structures have been determined of the pore-forming domains from colicins A and E1, along with the complete structures of colicins Ia, B and N [1–5]. The pore-forming domains are globular and contain ten a-helices (Figure 1a). Two hydrophobic helices lie at the core and form a hydrophobic helical hairpin that inserts into the membrane during the early stages of pore formation, by the mechanism described below.
Current Opinion in Structural Biology 2006, 16:230–236
Diphtheria toxin This review comes from a themed issue on Macromolecular assemblages Edited by Edward H Egelman and Andrew GW Leslie Available online 24th March 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.03.008
The pore-forming domain of diphtheria toxin also contains ten a-helices and includes a hydrophobic hairpin similar to that of the colicins [6] (Figure 1b). This a-PFT from the binary toxin family (A-B toxins) consists of two disulfide-linked chains resulting from protoxin cleavage. One chain contains the receptor-binding and pore-forming domains. The latter domain translocates the second chain, containing the catalytic domain, through the endosomal membrane to the cytosol, where it inhibits protein synthesis and kills the cell.
Introduction Bacterial toxins are some of the most potent poisons known to man. During invasion, bacterial toxins may inhibit a specific process, such as protein synthesis, or inflict a more general type of assault by punching holes in the membrane. The pore-forming toxins (PFTs) are ‘hole punchers’; the pores either deliver toxic components to specific cellular sites or kill cells through leakage. PFTs are interesting from several view points: many are virulence factors for disease, they challenge accepted ideas about protein sequence/structure relationships, and they also have potential as biotechnological sensors and delivery systems. PFTs are classified according to whether they form ahelical channels (a-PFTs) or b-barrels (b-PFTs). Here, we discuss recent examples of structural studies that are helping to unravel how these proteins transform from the soluble state to membrane-inserted pores.
Variations in the a-pore-forming domain
a-Pore-forming toxins
Membrane insertion
Colicins
Pore formation by a-PFTs requires three steps: binding, unfolding and insertion [9]. Binding, usually to a specific receptor, is followed by the unfolding of the pore-forming
Although the class of a-PFTs contains many major pathogens, the pore structures and even oligomeric states of Current Opinion in Structural Biology 2006, 16:230–236
Not all a-PFTs have an obvious hydrophobic hairpin. Despite structural similarity to both diphtheria and colicin pore-forming domains, the six a-helices that form the translocating pore of exotoxin A from Pseudomonas are not predominantly hydrophobic [7]. This is also true of the Bacillus thuringiensis Cry toxins, which cause osmotic lysis in the insect midgut. These proteins contain one domain that determines specificity, and two others that form the pore and modulate its properties. A recent structure of the mosquito-larvicidal toxin Cry4Ba demonstrates that only five of the seven a-helices in the pore-forming domain are necessary for toxicity [8]. This toxin is thought to form a membrane-inserting hairpin, as in colicins and diphtheria toxin, but the five amphipathic helices are arranged in a bundle with the most hydrophobic helix at the centre.
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Bacterial pore-formation toxins Tilley and Saibil 231
Figure 1
Structures of a-pore-forming domains. (a) Colicin B, (b) diphtheria toxin and (c) exotoxin A. Colicin B and diphtheria toxin anchor in the membrane using hydrophobic helical hairpins (red). In exotoxin A, the unfolding of helix F (orange) reveals the membrane anchor, Trp305 (red space-fill).
domain, which releases the hydrophobic helices that initiate insertion. For diphtheria toxin, which forms pores in the endosomal membrane, insertion of the hydrophobic hairpin is followed by insertion of the remaining poreforming helices [10]. In this case, acidification of the endosomes triggers unfolding. Recent evidence points to specific acidic residues that act as pH sensors in both the colicins and exotoxin A. A low pH environment disrupts a critical salt bridge and hydrogen-bonding network involving three conserved aspartate residues in colicin E1, causing local unfolding of a helix [11]. In exotoxin A, acidification neutralizes the charge on an aspartate residue at the end of a short length of helix, also triggering helix unfolding. This exposes a tryptophan residue that interacts with the membrane (Figure 1c), triggering insertion of the pore-forming domain [12]. In one case, the oligomeric state of an a-PFT has been determined. Monomers of the Cry1Ab toxin bind to one receptor, oligomerise into tetramers and then bind to a second receptor that targets the complex into membrane rafts, where the pores insert [13].
for small antimicrobial peptides [16]. In this model, the protein exerts a detergent-like action, inducing high curvature in the bilayer. Toroidal lipidic pores are characterized by their sensitivity to lipids that alter the curvature of the membrane, as is the case with colicin E1 pores [17]. Such a pore could be lined by shorter helices than those that would normally traverse the bilayer. Other a-pore-forming proteins
Further details on the a-PFT mechanism may come from sources other than bacterial toxins. The BCL-2 family of proteins regulates the apotoptic pathway and shares structural homology with the a-PFT pore-forming domains. They release mitochondrial apoptogenic factors by permeabilising the outer mitochondrial membrane (reviewed in [18]). Although the hydrophobic hairpin is present, it does not deeply insert and span the membrane [19]. Further work is needed before we can tell whether all these disparate features are snapshots of different intermediates, or whether a-PFTs follow different pathways and ultimately form different pores.
Unfolded intermediates and lipid structures
Intramolecular distance measurements on the partly inserted state suggest that the helices are loosely associated on the membrane surface, with the hydrophobic hairpin or aromatic residues inserted into the bilayer [14]. This was recently confirmed by measuring the inter- and intra-helical distances of colicin Ia by NMR [15]. However, the structure of the pore state is still very unclear. A model of the diphtheria toxin pore based on mutagenesis and fluorescence spectroscopy data supports the full insertion of the helical hairpin and suggests partial insertion of the remaining helices [10]. One problem with understanding the membrane-inserted form of the colicin pore is that the helices are too short to span a standard bilayer [9], although an increase in a-helical content indicates that they might lengthen before inserting [14]. A possible current model of the colicin pore is that of the toroidal lipidic pore, based on the model proposed www.sciencedirect.com
b-Pore-forming toxins
The insertion pathways and pore structures of b-PFTs are much better understood than those of the a-PFTs. b-PFTs are released as soluble monomers that oligomerise before forming active pores. The final insertion step often requires significant refolding of part of the structure, so that subunits can donate b-strands to form a transmembrane b-barrel. a-Hemolysin family
Named for their hemolytic activity, toxins from the Staphylococcus aureus hemolysin family form well-characterized pores. Members include heptameric a-hemolysin (a-HL) (Figure 2a) and the leukocidins, which form hetero-octamers of LukF and LukS subunits (Figure 2b,c) [20]. Like other b-PFTs, a-HL interacts directly with lipid vesicles, although it may bind to an Current Opinion in Structural Biology 2006, 16:230–236
232 Macromolecular assemblages
Figure 2
Structures of b-pore forming toxins. (a) a-HL subunit in the pore state, (b) LukF monomer, (c) LukS monomer, (d) aerolysin monomer, (e) ETX monomer, (f) anthrax PA63 from the heptameric prepore and (g) intermedilysin monomer. The regions that form the b-hairpins that insert into the membrane are coloured red.
unidentified receptor in vivo, possibly caveolin-1 [21]. a-HL oligomerises into a non-lytic heptamer on the membrane surface. This prepore state is a common feature of other b-PFTs and can be trapped with suitably engineered disulfide bonds [22]. The conformational changes that occur during membrane insertion have been identified by comparing the monomeric structures from the bicomponent leukocidin system with the pore state of a-HL (reviewed in [23]). The hemolysin subunit has three domains — the cap, rim and stem — with mainly b-structure. In the water-soluble state, the stem forms b-strands that fold against the monomer core [24]. To form the distinctive mushroom shape of the pore, the stem domains flip out and refold into b-hairpins to form a perfect transmembrane b-barrel [25] (Figure 3a,b). Aerolysin toxins
Aerolysin from Aeromonas hydrophila is an elongated fourdomain protein rich in b-structure [26] (Figure 2d). etoxin (ETX) from Clostridium perfringens is a structural homologue of aerolysin (Figure 2e) [27], whereas a-toxin (AT) from Clostridium septicum has been identified on the basis of high sequence homology. All three toxins are activated by protease cleavage, after which they bind to unidentified receptors, oligomerise and form heptameric pores [28–30]. However, aerolysin does not form a prepore before inserting, which is unusual for a b-PFT. The Current Opinion in Structural Biology 2006, 16:230–236
only structural model of the pore state for this family was created by docking the soluble aerolysin structure into a negative-stain EM map [26]. The pore-forming regions were identified by modelling AT based on the aerolysin structure. Fluorescence and biochemical techniques were used to confirm that they comprise a loop of 30 residues with alternating hydrophobic/hydrophilic character [31]. The equivalent region in the ETX structure is a loop containing a pair of b-strands (Figure 2e). Anthrax toxin
Like their a-PFT equivalents, b-PFT examples of binary toxins translocate toxic components into cells. The protective antigen (PA) from anthrax toxin forms the pore that delivers lethal factor and edema factor to the cytosol. Anthrax PA was first crystallized in soluble monomeric and heptameric forms [32] (Figures 2f and 3c,d), and structures of both forms have recently been determined in complex with their receptor [33,34]. Although the structure of the membrane-penetrating heptamer has not yet been solved, these structures provide many details about the mechanism of pore formation. PA has four domains and consists of mainly antiparallel b-sheets. Proteolytic cleavage of PA produces PA63, which oligomerises and inserts into the membrane. Because anthrax pores are formed in the endosomal membrane, it is no surprise that heptameric PA63 inserts at low pH [34], www.sciencedirect.com
Bacterial pore-formation toxins Tilley and Saibil 233
Figure 3
Oligomeric toxin structures. The a-HL pore viewed (a) from the side and (b) from the membrane. The b-barrel is formed from the b-hairpins (red). The hairpin-forming loop (red) of anthrax PA63 packs between subunits in the prepore state, as shown (c) from the side and (d) from the membrane.
unlike other b-PFTs. The membrane-spanning region has been identified as a loop that packs between monomers in the soluble heptameric structure [34]. In the pore form, this loop is thought to refold into a b-hairpin that is part of an unusually long, 14-stranded transmembrane b-barrel that extends 40 A˚ above the membrane surface. The resulting large gap is thought to be filled with the membrane-bound receptor [33]. Cholesterol-dependent cytolysins
Literally hole punchers, the cholesterol-dependent cytolysins (CDCs) form 250 A˚ pores comprising rings of 30– 50 subunits. They form a highly conserved family found in many Gram-positive bacteria. So far, crystal structures are available of the water-soluble monomers of perfringolysin and intermedilysin [35,36] (Figure 2g). These four-domain proteins are similar to members of the aerolysin family, sharing an elongated b-rich structure. Produced as soluble monomers or dimers [37], CDCs appear to bind to cholesterol, with the notable exception of intermedilysin, which binds the specific receptor CD59 [38]. After binding to the membrane surface, the CDCs form a well-characterized prepore state that can be trapped by mutagenesis or low temperature. Perfringolysin has been extensively probed using mutagenesis and spectroscopy to define the membranewww.sciencedirect.com
inserting regions. Whereas most b-PFTs donate one hairpin to the transmembrane b-barrel, CDCs donate two [39,40], creating large pores that leak macromolecules and smaller solutes. Although all PFTs rearrange their structure to form the pore, these toxins are the only example known to switch secondary structure. Each bhairpin originates from an a-helical bundle in the soluble monomer. Before insertion can occur, this region must be brought to the membrane surface. We recently obtained electron density maps of prepores and pores of the CDC pneumolysin, which is closely related to perfringolysin, using cryo-EM and docking of the perfringolysin domains [41]. In the prepore, the subunits resemble the extended soluble monomer, with the pore-forming regions far above the membrane surface. In the pore structure, the domains separate, and the domain containing the transmembrane region swings out and is brought 30 A˚ closer to the membrane by the collapse of another domain, forming an arch structure (Figure 4). A similar height decrease of the protein above the membrane surface was observed in the perfringolysin prepore-to-pore conversion by atomic force microscopy [42]. In the cryoEM reconstruction, the membrane is deformed by its interaction with the protein and the pore lumen is completely empty, apparently after extrusion of the missing disk of membrane. The pore is lined by protein density consistent with the length of b-hairpins extending across Current Opinion in Structural Biology 2006, 16:230–236
234 Macromolecular assemblages
Figure 4
The pneumolysin pore. (a) Cryo-EM reconstruction of the pneumolysin pore with 38 subunits. The inset shows an arc of subunits in the pore conformation docked into the density. (b) Vertical cross-section through the pore, showing the perfringolysin domains (cyan) docked into the pore density, with the b-hairpins (red) extending through the bilayer.
the bilayer to form the pore wall, which comprises a b-barrel with up to 176 strands (Figure 4). What triggers insertion into the membrane? Oligomerisation in the prepore causes changes in the domain packing arrangement and may help to trigger the subsequent opening up of the cavity in the pore conformation [41]. Both cholesterol and a conserved tryptophan-rich motif in the membrane-binding domain are involved in the prepore-to-pore conversion [36,43], suggesting that cholesterol does more than act as a receptor for CDCs. Cholesterol may fill the interstitial spaces around the protein to allow an unusual protein–lipid interaction in which the arch of protein effects a 308 bend in the bilayer [41] (Figure 4b).
are able to insert spontaneously, more like b-barrels. There are still many issues to be understood concerning the interaction between toxin and membrane. A general strategy appears to be that pore regions are buried or pack against the surface of the soluble structure, and then exchange their protein–protein interactions for protein– lipid interactions in a stable new assembly. The protein may act by inserting a wedge into the membrane, changing its curvature and ultimately puncturing the bilayer to form a stable pore lining.
Acknowledgments We thank Ambrose Cole for comments on the manuscript, and the UK Biotechnology and Biological Sciences Research Council for financial support.
References and recommended reading Eukaryotic b-pore-forming toxins
Many of these toxin families have been thought to be unique to bacteria, but structural homologues are now being discovered in eukaryotes. Enterolobin from the Brazilian Enterolobium contortiliquum tree, a toxic lectin from the mushroom Laetiporus sulphureus and a family of hydralysins from Cnidaria belong to the aerolysin-like toxin family [44–46], whereas fungus volvatoxin A2 is a homologue of Cyt d-endotoxin [47].
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Wiener M, Freymann D, Ghosh P, Stroud RM: Crystal structure of colicin Ia. Nature 1997, 385:461-464.
2.
Vetter IR, Parker MW, Tucker AD, Lakey JH, Pattus F, Tsernoglou D: Crystal structure of a colicin N fragment suggests a model for toxicity. Structure 1998, 6:863-874.
3.
Hilsenbeck JL, Park H, Chen G, Youn B, Postle K, Kang C: Crystal structure of the cytotoxic bacterial protein colicin B at 2.5 A˚ resolution. Mol Microbiol 2004, 51:711-720.
4.
Parker MW, Postma JP, Pattus F, Tucker AD, Tsernoglou D: Refined structure of the pore-forming domain of colicin A at 2.4 A˚ resolution. J Mol Biol 1992, 224:639-657.
5.
Elkins P, Bunker A, Cramer WA, Stauffacher CV: A mechanism for toxin insertion into membranes is suggested by the crystal structure of the channel-forming domain of colicin E1. Structure 1997, 5:443-458.
6.
Bennett MJ, Choe S, Eisenberg D: Refined structure of dimeric diphtheria toxin at 2.0 A˚ resolution. Protein Sci 1994, 3:1444-1463.
Conclusions As multiple structures of toxin family members are identified, structural comparisons help us to identify common motifs and design experiments that probe their poreforming mechanisms. Molecular dynamics simulations have been used to model pore formation and membrane rupture in lipid bilayers [48]. However, many aspects of the protein structural transformations are still obscure. In particular, a-helical membrane proteins are normally inserted by the Sec61 translocon machinery, but a-PFTs Current Opinion in Structural Biology 2006, 16:230–236
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Bacterial pore-formation toxins Tilley and Saibil 235
7.
Allured VS, Collier RJ, Carroll SF, McKay DB: Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc Natl Acad Sci USA 1986, 83:1320-1324.
8.
Boonserm P, Davis P, Ellar DJ, Li J: Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications. J Mol Biol 2005, 348:363-382.
9.
Zakharov SD, Kotova EA, Antonenko YN, Cramer WA: On the role of lipid in colicin pore formation. Biochim Biophys Acta 2004, 1666:239-249.
10. Zhao G, London E: Behavior of diphtheria toxin T domain containing substitutions that block normal membrane insertion at Pro345 and Leu307: control of deep membrane insertion and coupling between deep insertion of hydrophobic subdomains. Biochemistry 2005, 44:4488-4498. 11. Musse AA, Merrill AR: The molecular basis for the pH-activation mechanism in the channel-forming bacterial colicin E1. J Biol Chem 2003, 278:24491-24499. 12. Mere J, Morlon-Guyot J, Bonhoure A, Chiche L, Beaumelle B: Acid-triggered membrane insertion of Pseudomonas exotoxin A involves an original mechanism based on pH-regulated tryptophan exposure. J Biol Chem 2005, 280:21194-21201. 13. Bravo A, Gomez I, Conde J, Munoz-Garay C, Sanchez J, Miranda R, Zhuang M, Gill SS, Soberon M: Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab poreforming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim Biophys Acta 2004, 1667:38-46. 14. Zakharov SD, Lindeberg M, Griko Y, Salamon Z, Tollin G, Prendergast FG, Cramer WA: Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array. Proc Natl Acad Sci USA 1998, 95:4282-4287. 15. Luo W, Yao X, Hong M: Large structure rearrangement of colicin Ia channel domain after membrane binding from 2D 13C spin diffusion NMR. J Am Chem Soc 2005, 127:6402-6408. 16. Zasloff M: Antimicrobial peptides of multicellular organisms. Nature 2002, 415:389-395. 17. Sobko AA, Kotova EA, Antonenko YN, Zakharov SD, Cramer WA: Effect of lipids with different spontaneous curvature on the channel activity of colicin E1: evidence in favor of a toroidal pore. FEBS Lett 2004, 576:205-210. 18. Breckenridge DG, Xue D: Regulation of mitochondrial membrane permeabilization by BCL-2 family proteins and caspases. Curr Opin Cell Biol 2004, 16:647-652. 19. Oh KJ, Barbuto S, Meyer N, Kim RS, Collier RJ, Korsmeyer SJ: Conformational changes in BID, a pro-apoptotic BCL-2 family member, upon membrane binding, A site-directed spin labeling study. J Biol Chem 2005, 280:753-767. 20. Jayasinghe L, Bayley H: The leukocidin pore: evidence for an octamer with four LukF subunits and four LukS subunits alternating around a central axis. Protein Sci 2005, 14:2550-2561. 21. Pany S, Vijayvargia R, Krishnasastry MV: Caveolin-1 binding motif of alpha-hemolysin: its role in stability and pore formation. Biochem Biophys Res Commun 2004, 322:29-36. 22. Kawate T, Gouaux E: Arresting and releasing Staphylococcal alpha-hemolysin at intermediate stages of pore formation by engineered disulfide bonds. Protein Sci 2003, 12:997-1006. 23. Menestrina G, Dalla Serra M, Comai M, Coraiola M, Viero G, Werner S, Colin DA, Monteil H, Prevost G: Ion channels and bacterial infection: the case of beta-barrel pore-forming protein toxins of Staphylococcus aureus. FEBS Lett 2003, 552:54-60. 24. Guillet V, Roblin P, Werner S, Coraiola M, Menestrina G, Monteil H, Prevost G, Mourey L: Crystal structure of leucotoxin S component: new insight into the Staphylococcal beta-barrel pore-forming toxins. J Biol Chem 2004, 279:41028-41037. 25. Song L, Hobaugh MR, Shustak C, Cheley S, Bayley H, Gouaux JE: Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 1996, 274:1859-1866. www.sciencedirect.com
26. Parker MW, Buckley JT, Postma JP, Tucker AD, Leonard K, Pattus F, Tsernoglou D: Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states. Nature 1994, 367:292-295. 27. Cole AR, Gibert M, Popoff M, Moss DS, Titball RW, Basak AK: Clostridium perfringens epsilon-toxin shows structural similarity to the pore-forming toxin aerolysin. Nat Struct Mol Biol 2004, 11:797-798. ETX is a structural homologue of aerolysin, despite minimal sequence similarity. This study confirms the presence of a loop that is thought to form the amphipathic b-hairpin. 28. Moniatte M, van der Goot FG, Buckley JT, Pattus F, van Dorsselaer A: Characterisation of the heptameric pore-forming complex of the Aeromonas toxin aerolysin using MALDI-TOF mass spectrometry. FEBS Lett 1996, 384:269-272. 29. Krasilnikov OV, Merzlyak PG, Yuldasheva LN, Rodrigues CG, Bhakdi S, Valeva A: Electrophysiological evidence for heptameric stoichiometry of ion channels formed by Staphylococcus aureus alpha-toxin in planar lipid bilayers. Mol Microbiol 2000, 37:1372-1378. 30. Miyata S, Matsushita O, Minami J, Katayama S, Shimamoto S, Okabe A: Cleavage of a C-terminal peptide is essential for heptamerization of Clostridium perfringens epsilon-toxin in the synaptosomal membrane. J Biol Chem 2001, 276:13778-13783. 31. Melton JA, Parker MW, Rossjohn J, Buckley JT, Tweten RK: The identification and structure of the membrane-spanning domain of the Clostridium septicum alpha toxin. J Biol Chem 2004, 279:14315-14322. The membrane-penetrating region was identified from the model of AT based on the aerolysin structure. Fluorescence and mutagenesis were used to confirm that a loop forms the amphipathic b-hairpin, providing the first experimental data on the pore-forming region of the aerolysin family. 32. Petosa C, Collier RJ, Klimpel KR, Leppla SH, Liddington RC: Crystal structure of the anthrax toxin protective antigen. Nature 1997, 385:833-838. 33. Santelli E, Bankston LA, Leppla SH, Liddington RC: Crystal structure of a complex between anthrax toxin and its host cell receptor. Nature 2004, 430:905-908. 34. Lacy DB, Wigelsworth DJ, Melnyk RA, Harrison SC, Collier RJ: Structure of heptameric protective antigen bound to an anthrax toxin receptor: a role for receptor in pH-dependent pore formation. Proc Natl Acad Sci USA 2004, 101:13147-13151. The authors present structures of the anthrax PA prepore at 3.6 A˚ and the prepore–receptor complex at 4.3 A˚. This shows, for the first time, the position of the pore-forming loops that form the b-hairpin and also that the receptor binds to both domains 2 and 4, indicating that this interaction prevents premature formation of the pore. 35. Rossjohn J, Feil SC, McKinstry WJ, Tweten RK, Parker MW: Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form. Cell 1997, 89:685-692. 36. Polekhina G, Giddings KS, Tweten RK, Parker MW: Insights into the action of the superfamily of cholesterol-dependent cytolysins from studies of intermedilysin. Proc Natl Acad Sci USA 2005, 102:600-605. The second structure of a CDC, intermedilysin, confirms the highly conserved structure of this family, and indicates that the link between domain 4 and the remainder of the protein is flexible. Mutagenesis of the conserved tryptophan motif locks the toxin in the prepore state. 37. Solovyova AS, Nollmann M, Mitchell TJ, Byron O: The solution structure and oligomerization behavior of two bacterial toxins: pneumolysin and perfringolysin O. Biophys J 2004, 87:540-552. 38. Giddings KS, Zhao J, Sims PJ, Tweten RK: Human CD59 is a receptor for the cholesterol-dependent cytolysin intermedilysin. Nat Struct Mol Biol 2004, 11:1173-1178. 39. Shepard LA, Heuck AP, Hamman BD, Rossjohn J, Parker MW, Ryan KR, Johnson AE, Tweten RK: Identification of a membrane-spanning domain of the thiol-activated poreforming toxin Clostridium perfringens perfringolysin O: an a-helical to b-sheet transition identified by fluorescence spectroscopy. Biochemistry 1998, 37:14563-14574. Current Opinion in Structural Biology 2006, 16:230–236
236 Macromolecular assemblages
40. Shatursky O, Heuck AP, Shepard LA, Rossjohn J, Parker MW, Johnson AE, Tweten RK: The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins. Cell 1999, 99:293-299.
44. Bittencourt SE, Silva LP, Azevedo RB, Cunha RB, Lima CM, Ricart CA, Sousa MV: The plant cytolytic protein enterolobin assumes a dimeric structure in solution. FEBS Lett 2003, 549:47-51.
41. Tilley SJ, Orlova EV, Gilbert RJ, Andrew PW, Saibil HR: Structural basis of pore formation by the bacterial toxin pneumolysin. Cell 2005, 121:247-256. Fitting perfringolysin into cryo-EM maps of the prepore and pore states of pneumolysin shows that the toxin domains separate and that domain 2 collapses down onto the membrane, allowing insertion of the b-hairpins to form the pore wall.
45. Mancheno JM, Tateno H, Goldstein IJ, Martinez-Ripoll M, Hermoso JA: Structural analysis of the Laetiporus sulphureus hemolytic pore-forming lectin in complex with sugars. J Biol Chem 2005, 280:17251-17259.
42. Czajkowsky DM, Hotze EM, Shao Z, Tweten RK: Vertical collapse of a cytolysin prepore moves its transmembrane b-hairpins to the membrane. EMBO J 2004, 23:3206-3215. Atomic force microscopy measurements show that the prepore-to-pore conversion of perfringolysin is accompanied by a height reduction of 40 A˚. 43. Giddings KS, Johnson AE, Tweten RK: Redefining cholesterol’s role in the mechanism of the cholesterol-dependent cytolysins. Proc Natl Acad Sci USA 2003, 100:11315-11320.
Current Opinion in Structural Biology 2006, 16:230–236
46. Sher D, Fishman Y, Zhang M, Lebendiker M, Gaathon A, Mancheno JM, Zlotkin E: Hydralysins, a new category of beta-pore-forming toxins in cnidaria. J Biol Chem 2005, 280:22847-22855. 47. Lin SC, Lo YC, Lin JY, Liaw YC: Crystal structures and electron micrographs of fungal volvatoxin A2. J Mol Biol 2004, 343:477-491. 48. Tieleman DP, Leontiadou H, Mark AE, Marrink SJ: Simulation of pore formation in lipid bilayers by mechanical stress and electric fields. J Am Chem Soc 2003, 125:6382-6383.
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members of this family are still very unclear. The bacterial toxin colicin targets Escherichia coli cells by making pores that leak ions, depolarizing the membrane and ultimately causing cell death. Colicins usually contain three domains that are involved in receptor binding, transport and killing. Depending on the colicin, the toxic (killing) domain either inhibits protein or peptidoglycan synthesis, or has nuclease or pore-forming activity. The other domains are used to bind and translocate the toxic domain into the bacterial cell. Structures have been determined of the pore-forming domains from colicins A and E1, along with the complete structures of colicins Ia, B and N [1–5]. The pore-forming domains are globular and contain ten a-helices (Figure 1a). Two hydrophobic helices lie at the core and form a hydrophobic helical hairpin that inserts into the membrane during the early stages of pore formation, by the mechanism described below.
Current Opinion in Structural Biology 2006, 16:230–236
Diphtheria toxin This review comes from a themed issue on Macromolecular assemblages Edited by Edward H Egelman and Andrew GW Leslie Available online 24th March 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.03.008
The pore-forming domain of diphtheria toxin also contains ten a-helices and includes a hydrophobic hairpin similar to that of the colicins [6] (Figure 1b). This a-PFT from the binary toxin family (A-B toxins) consists of two disulfide-linked chains resulting from protoxin cleavage. One chain contains the receptor-binding and pore-forming domains. The latter domain translocates the second chain, containing the catalytic domain, through the endosomal membrane to the cytosol, where it inhibits protein synthesis and kills the cell.
Introduction Bacterial toxins are some of the most potent poisons known to man. During invasion, bacterial toxins may inhibit a specific process, such as protein synthesis, or inflict a more general type of assault by punching holes in the membrane. The pore-forming toxins (PFTs) are ‘hole punchers’; the pores either deliver toxic components to specific cellular sites or kill cells through leakage. PFTs are interesting from several view points: many are virulence factors for disease, they challenge accepted ideas about protein sequence/structure relationships, and they also have potential as biotechnological sensors and delivery systems. PFTs are classified according to whether they form ahelical channels (a-PFTs) or b-barrels (b-PFTs). Here, we discuss recent examples of structural studies that are helping to unravel how these proteins transform from the soluble state to membrane-inserted pores.
Variations in the a-pore-forming domain
a-Pore-forming toxins
Membrane insertion
Colicins
Pore formation by a-PFTs requires three steps: binding, unfolding and insertion [9]. Binding, usually to a specific receptor, is followed by the unfolding of the pore-forming
Although the class of a-PFTs contains many major pathogens, the pore structures and even oligomeric states of Current Opinion in Structural Biology 2006, 16:230–236
Not all a-PFTs have an obvious hydrophobic hairpin. Despite structural similarity to both diphtheria and colicin pore-forming domains, the six a-helices that form the translocating pore of exotoxin A from Pseudomonas are not predominantly hydrophobic [7]. This is also true of the Bacillus thuringiensis Cry toxins, which cause osmotic lysis in the insect midgut. These proteins contain one domain that determines specificity, and two others that form the pore and modulate its properties. A recent structure of the mosquito-larvicidal toxin Cry4Ba demonstrates that only five of the seven a-helices in the pore-forming domain are necessary for toxicity [8]. This toxin is thought to form a membrane-inserting hairpin, as in colicins and diphtheria toxin, but the five amphipathic helices are arranged in a bundle with the most hydrophobic helix at the centre.
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Bacterial pore-formation toxins Tilley and Saibil 231
Figure 1
Structures of a-pore-forming domains. (a) Colicin B, (b) diphtheria toxin and (c) exotoxin A. Colicin B and diphtheria toxin anchor in the membrane using hydrophobic helical hairpins (red). In exotoxin A, the unfolding of helix F (orange) reveals the membrane anchor, Trp305 (red space-fill).
domain, which releases the hydrophobic helices that initiate insertion. For diphtheria toxin, which forms pores in the endosomal membrane, insertion of the hydrophobic hairpin is followed by insertion of the remaining poreforming helices [10]. In this case, acidification of the endosomes triggers unfolding. Recent evidence points to specific acidic residues that act as pH sensors in both the colicins and exotoxin A. A low pH environment disrupts a critical salt bridge and hydrogen-bonding network involving three conserved aspartate residues in colicin E1, causing local unfolding of a helix [11]. In exotoxin A, acidification neutralizes the charge on an aspartate residue at the end of a short length of helix, also triggering helix unfolding. This exposes a tryptophan residue that interacts with the membrane (Figure 1c), triggering insertion of the pore-forming domain [12]. In one case, the oligomeric state of an a-PFT has been determined. Monomers of the Cry1Ab toxin bind to one receptor, oligomerise into tetramers and then bind to a second receptor that targets the complex into membrane rafts, where the pores insert [13].
for small antimicrobial peptides [16]. In this model, the protein exerts a detergent-like action, inducing high curvature in the bilayer. Toroidal lipidic pores are characterized by their sensitivity to lipids that alter the curvature of the membrane, as is the case with colicin E1 pores [17]. Such a pore could be lined by shorter helices than those that would normally traverse the bilayer. Other a-pore-forming proteins
Further details on the a-PFT mechanism may come from sources other than bacterial toxins. The BCL-2 family of proteins regulates the apotoptic pathway and shares structural homology with the a-PFT pore-forming domains. They release mitochondrial apoptogenic factors by permeabilising the outer mitochondrial membrane (reviewed in [18]). Although the hydrophobic hairpin is present, it does not deeply insert and span the membrane [19]. Further work is needed before we can tell whether all these disparate features are snapshots of different intermediates, or whether a-PFTs follow different pathways and ultimately form different pores.
Unfolded intermediates and lipid structures
Intramolecular distance measurements on the partly inserted state suggest that the helices are loosely associated on the membrane surface, with the hydrophobic hairpin or aromatic residues inserted into the bilayer [14]. This was recently confirmed by measuring the inter- and intra-helical distances of colicin Ia by NMR [15]. However, the structure of the pore state is still very unclear. A model of the diphtheria toxin pore based on mutagenesis and fluorescence spectroscopy data supports the full insertion of the helical hairpin and suggests partial insertion of the remaining helices [10]. One problem with understanding the membrane-inserted form of the colicin pore is that the helices are too short to span a standard bilayer [9], although an increase in a-helical content indicates that they might lengthen before inserting [14]. A possible current model of the colicin pore is that of the toroidal lipidic pore, based on the model proposed www.sciencedirect.com
b-Pore-forming toxins
The insertion pathways and pore structures of b-PFTs are much better understood than those of the a-PFTs. b-PFTs are released as soluble monomers that oligomerise before forming active pores. The final insertion step often requires significant refolding of part of the structure, so that subunits can donate b-strands to form a transmembrane b-barrel. a-Hemolysin family
Named for their hemolytic activity, toxins from the Staphylococcus aureus hemolysin family form well-characterized pores. Members include heptameric a-hemolysin (a-HL) (Figure 2a) and the leukocidins, which form hetero-octamers of LukF and LukS subunits (Figure 2b,c) [20]. Like other b-PFTs, a-HL interacts directly with lipid vesicles, although it may bind to an Current Opinion in Structural Biology 2006, 16:230–236
232 Macromolecular assemblages
Figure 2
Structures of b-pore forming toxins. (a) a-HL subunit in the pore state, (b) LukF monomer, (c) LukS monomer, (d) aerolysin monomer, (e) ETX monomer, (f) anthrax PA63 from the heptameric prepore and (g) intermedilysin monomer. The regions that form the b-hairpins that insert into the membrane are coloured red.
unidentified receptor in vivo, possibly caveolin-1 [21]. a-HL oligomerises into a non-lytic heptamer on the membrane surface. This prepore state is a common feature of other b-PFTs and can be trapped with suitably engineered disulfide bonds [22]. The conformational changes that occur during membrane insertion have been identified by comparing the monomeric structures from the bicomponent leukocidin system with the pore state of a-HL (reviewed in [23]). The hemolysin subunit has three domains — the cap, rim and stem — with mainly b-structure. In the water-soluble state, the stem forms b-strands that fold against the monomer core [24]. To form the distinctive mushroom shape of the pore, the stem domains flip out and refold into b-hairpins to form a perfect transmembrane b-barrel [25] (Figure 3a,b). Aerolysin toxins
Aerolysin from Aeromonas hydrophila is an elongated fourdomain protein rich in b-structure [26] (Figure 2d). etoxin (ETX) from Clostridium perfringens is a structural homologue of aerolysin (Figure 2e) [27], whereas a-toxin (AT) from Clostridium septicum has been identified on the basis of high sequence homology. All three toxins are activated by protease cleavage, after which they bind to unidentified receptors, oligomerise and form heptameric pores [28–30]. However, aerolysin does not form a prepore before inserting, which is unusual for a b-PFT. The Current Opinion in Structural Biology 2006, 16:230–236
only structural model of the pore state for this family was created by docking the soluble aerolysin structure into a negative-stain EM map [26]. The pore-forming regions were identified by modelling AT based on the aerolysin structure. Fluorescence and biochemical techniques were used to confirm that they comprise a loop of 30 residues with alternating hydrophobic/hydrophilic character [31]. The equivalent region in the ETX structure is a loop containing a pair of b-strands (Figure 2e). Anthrax toxin
Like their a-PFT equivalents, b-PFT examples of binary toxins translocate toxic components into cells. The protective antigen (PA) from anthrax toxin forms the pore that delivers lethal factor and edema factor to the cytosol. Anthrax PA was first crystallized in soluble monomeric and heptameric forms [32] (Figures 2f and 3c,d), and structures of both forms have recently been determined in complex with their receptor [33,34]. Although the structure of the membrane-penetrating heptamer has not yet been solved, these structures provide many details about the mechanism of pore formation. PA has four domains and consists of mainly antiparallel b-sheets. Proteolytic cleavage of PA produces PA63, which oligomerises and inserts into the membrane. Because anthrax pores are formed in the endosomal membrane, it is no surprise that heptameric PA63 inserts at low pH [34], www.sciencedirect.com
Bacterial pore-formation toxins Tilley and Saibil 233
Figure 3
Oligomeric toxin structures. The a-HL pore viewed (a) from the side and (b) from the membrane. The b-barrel is formed from the b-hairpins (red). The hairpin-forming loop (red) of anthrax PA63 packs between subunits in the prepore state, as shown (c) from the side and (d) from the membrane.
unlike other b-PFTs. The membrane-spanning region has been identified as a loop that packs between monomers in the soluble heptameric structure [34]. In the pore form, this loop is thought to refold into a b-hairpin that is part of an unusually long, 14-stranded transmembrane b-barrel that extends 40 A˚ above the membrane surface. The resulting large gap is thought to be filled with the membrane-bound receptor [33]. Cholesterol-dependent cytolysins
Literally hole punchers, the cholesterol-dependent cytolysins (CDCs) form 250 A˚ pores comprising rings of 30– 50 subunits. They form a highly conserved family found in many Gram-positive bacteria. So far, crystal structures are available of the water-soluble monomers of perfringolysin and intermedilysin [35,36] (Figure 2g). These four-domain proteins are similar to members of the aerolysin family, sharing an elongated b-rich structure. Produced as soluble monomers or dimers [37], CDCs appear to bind to cholesterol, with the notable exception of intermedilysin, which binds the specific receptor CD59 [38]. After binding to the membrane surface, the CDCs form a well-characterized prepore state that can be trapped by mutagenesis or low temperature. Perfringolysin has been extensively probed using mutagenesis and spectroscopy to define the membranewww.sciencedirect.com
inserting regions. Whereas most b-PFTs donate one hairpin to the transmembrane b-barrel, CDCs donate two [39,40], creating large pores that leak macromolecules and smaller solutes. Although all PFTs rearrange their structure to form the pore, these toxins are the only example known to switch secondary structure. Each bhairpin originates from an a-helical bundle in the soluble monomer. Before insertion can occur, this region must be brought to the membrane surface. We recently obtained electron density maps of prepores and pores of the CDC pneumolysin, which is closely related to perfringolysin, using cryo-EM and docking of the perfringolysin domains [41]. In the prepore, the subunits resemble the extended soluble monomer, with the pore-forming regions far above the membrane surface. In the pore structure, the domains separate, and the domain containing the transmembrane region swings out and is brought 30 A˚ closer to the membrane by the collapse of another domain, forming an arch structure (Figure 4). A similar height decrease of the protein above the membrane surface was observed in the perfringolysin prepore-to-pore conversion by atomic force microscopy [42]. In the cryoEM reconstruction, the membrane is deformed by its interaction with the protein and the pore lumen is completely empty, apparently after extrusion of the missing disk of membrane. The pore is lined by protein density consistent with the length of b-hairpins extending across Current Opinion in Structural Biology 2006, 16:230–236
234 Macromolecular assemblages
Figure 4
The pneumolysin pore. (a) Cryo-EM reconstruction of the pneumolysin pore with 38 subunits. The inset shows an arc of subunits in the pore conformation docked into the density. (b) Vertical cross-section through the pore, showing the perfringolysin domains (cyan) docked into the pore density, with the b-hairpins (red) extending through the bilayer.
the bilayer to form the pore wall, which comprises a b-barrel with up to 176 strands (Figure 4). What triggers insertion into the membrane? Oligomerisation in the prepore causes changes in the domain packing arrangement and may help to trigger the subsequent opening up of the cavity in the pore conformation [41]. Both cholesterol and a conserved tryptophan-rich motif in the membrane-binding domain are involved in the prepore-to-pore conversion [36,43], suggesting that cholesterol does more than act as a receptor for CDCs. Cholesterol may fill the interstitial spaces around the protein to allow an unusual protein–lipid interaction in which the arch of protein effects a 308 bend in the bilayer [41] (Figure 4b).
are able to insert spontaneously, more like b-barrels. There are still many issues to be understood concerning the interaction between toxin and membrane. A general strategy appears to be that pore regions are buried or pack against the surface of the soluble structure, and then exchange their protein–protein interactions for protein– lipid interactions in a stable new assembly. The protein may act by inserting a wedge into the membrane, changing its curvature and ultimately puncturing the bilayer to form a stable pore lining.
Acknowledgments We thank Ambrose Cole for comments on the manuscript, and the UK Biotechnology and Biological Sciences Research Council for financial support.
References and recommended reading Eukaryotic b-pore-forming toxins
Many of these toxin families have been thought to be unique to bacteria, but structural homologues are now being discovered in eukaryotes. Enterolobin from the Brazilian Enterolobium contortiliquum tree, a toxic lectin from the mushroom Laetiporus sulphureus and a family of hydralysins from Cnidaria belong to the aerolysin-like toxin family [44–46], whereas fungus volvatoxin A2 is a homologue of Cyt d-endotoxin [47].
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Wiener M, Freymann D, Ghosh P, Stroud RM: Crystal structure of colicin Ia. Nature 1997, 385:461-464.
2.
Vetter IR, Parker MW, Tucker AD, Lakey JH, Pattus F, Tsernoglou D: Crystal structure of a colicin N fragment suggests a model for toxicity. Structure 1998, 6:863-874.
3.
Hilsenbeck JL, Park H, Chen G, Youn B, Postle K, Kang C: Crystal structure of the cytotoxic bacterial protein colicin B at 2.5 A˚ resolution. Mol Microbiol 2004, 51:711-720.
4.
Parker MW, Postma JP, Pattus F, Tucker AD, Tsernoglou D: Refined structure of the pore-forming domain of colicin A at 2.4 A˚ resolution. J Mol Biol 1992, 224:639-657.
5.
Elkins P, Bunker A, Cramer WA, Stauffacher CV: A mechanism for toxin insertion into membranes is suggested by the crystal structure of the channel-forming domain of colicin E1. Structure 1997, 5:443-458.
6.
Bennett MJ, Choe S, Eisenberg D: Refined structure of dimeric diphtheria toxin at 2.0 A˚ resolution. Protein Sci 1994, 3:1444-1463.
Conclusions As multiple structures of toxin family members are identified, structural comparisons help us to identify common motifs and design experiments that probe their poreforming mechanisms. Molecular dynamics simulations have been used to model pore formation and membrane rupture in lipid bilayers [48]. However, many aspects of the protein structural transformations are still obscure. In particular, a-helical membrane proteins are normally inserted by the Sec61 translocon machinery, but a-PFTs Current Opinion in Structural Biology 2006, 16:230–236
www.sciencedirect.com
Bacterial pore-formation toxins Tilley and Saibil 235
7.
Allured VS, Collier RJ, Carroll SF, McKay DB: Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc Natl Acad Sci USA 1986, 83:1320-1324.
8.
Boonserm P, Davis P, Ellar DJ, Li J: Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications. J Mol Biol 2005, 348:363-382.
9.
Zakharov SD, Kotova EA, Antonenko YN, Cramer WA: On the role of lipid in colicin pore formation. Biochim Biophys Acta 2004, 1666:239-249.
10. Zhao G, London E: Behavior of diphtheria toxin T domain containing substitutions that block normal membrane insertion at Pro345 and Leu307: control of deep membrane insertion and coupling between deep insertion of hydrophobic subdomains. Biochemistry 2005, 44:4488-4498. 11. Musse AA, Merrill AR: The molecular basis for the pH-activation mechanism in the channel-forming bacterial colicin E1. J Biol Chem 2003, 278:24491-24499. 12. Mere J, Morlon-Guyot J, Bonhoure A, Chiche L, Beaumelle B: Acid-triggered membrane insertion of Pseudomonas exotoxin A involves an original mechanism based on pH-regulated tryptophan exposure. J Biol Chem 2005, 280:21194-21201. 13. Bravo A, Gomez I, Conde J, Munoz-Garay C, Sanchez J, Miranda R, Zhuang M, Gill SS, Soberon M: Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab poreforming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim Biophys Acta 2004, 1667:38-46. 14. Zakharov SD, Lindeberg M, Griko Y, Salamon Z, Tollin G, Prendergast FG, Cramer WA: Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array. Proc Natl Acad Sci USA 1998, 95:4282-4287. 15. Luo W, Yao X, Hong M: Large structure rearrangement of colicin Ia channel domain after membrane binding from 2D 13C spin diffusion NMR. J Am Chem Soc 2005, 127:6402-6408. 16. Zasloff M: Antimicrobial peptides of multicellular organisms. Nature 2002, 415:389-395. 17. Sobko AA, Kotova EA, Antonenko YN, Zakharov SD, Cramer WA: Effect of lipids with different spontaneous curvature on the channel activity of colicin E1: evidence in favor of a toroidal pore. FEBS Lett 2004, 576:205-210. 18. Breckenridge DG, Xue D: Regulation of mitochondrial membrane permeabilization by BCL-2 family proteins and caspases. Curr Opin Cell Biol 2004, 16:647-652. 19. Oh KJ, Barbuto S, Meyer N, Kim RS, Collier RJ, Korsmeyer SJ: Conformational changes in BID, a pro-apoptotic BCL-2 family member, upon membrane binding, A site-directed spin labeling study. J Biol Chem 2005, 280:753-767. 20. Jayasinghe L, Bayley H: The leukocidin pore: evidence for an octamer with four LukF subunits and four LukS subunits alternating around a central axis. Protein Sci 2005, 14:2550-2561. 21. Pany S, Vijayvargia R, Krishnasastry MV: Caveolin-1 binding motif of alpha-hemolysin: its role in stability and pore formation. Biochem Biophys Res Commun 2004, 322:29-36. 22. Kawate T, Gouaux E: Arresting and releasing Staphylococcal alpha-hemolysin at intermediate stages of pore formation by engineered disulfide bonds. Protein Sci 2003, 12:997-1006. 23. Menestrina G, Dalla Serra M, Comai M, Coraiola M, Viero G, Werner S, Colin DA, Monteil H, Prevost G: Ion channels and bacterial infection: the case of beta-barrel pore-forming protein toxins of Staphylococcus aureus. FEBS Lett 2003, 552:54-60. 24. Guillet V, Roblin P, Werner S, Coraiola M, Menestrina G, Monteil H, Prevost G, Mourey L: Crystal structure of leucotoxin S component: new insight into the Staphylococcal beta-barrel pore-forming toxins. J Biol Chem 2004, 279:41028-41037. 25. Song L, Hobaugh MR, Shustak C, Cheley S, Bayley H, Gouaux JE: Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 1996, 274:1859-1866. www.sciencedirect.com
26. Parker MW, Buckley JT, Postma JP, Tucker AD, Leonard K, Pattus F, Tsernoglou D: Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states. Nature 1994, 367:292-295. 27. Cole AR, Gibert M, Popoff M, Moss DS, Titball RW, Basak AK: Clostridium perfringens epsilon-toxin shows structural similarity to the pore-forming toxin aerolysin. Nat Struct Mol Biol 2004, 11:797-798. ETX is a structural homologue of aerolysin, despite minimal sequence similarity. This study confirms the presence of a loop that is thought to form the amphipathic b-hairpin. 28. Moniatte M, van der Goot FG, Buckley JT, Pattus F, van Dorsselaer A: Characterisation of the heptameric pore-forming complex of the Aeromonas toxin aerolysin using MALDI-TOF mass spectrometry. FEBS Lett 1996, 384:269-272. 29. Krasilnikov OV, Merzlyak PG, Yuldasheva LN, Rodrigues CG, Bhakdi S, Valeva A: Electrophysiological evidence for heptameric stoichiometry of ion channels formed by Staphylococcus aureus alpha-toxin in planar lipid bilayers. Mol Microbiol 2000, 37:1372-1378. 30. Miyata S, Matsushita O, Minami J, Katayama S, Shimamoto S, Okabe A: Cleavage of a C-terminal peptide is essential for heptamerization of Clostridium perfringens epsilon-toxin in the synaptosomal membrane. J Biol Chem 2001, 276:13778-13783. 31. Melton JA, Parker MW, Rossjohn J, Buckley JT, Tweten RK: The identification and structure of the membrane-spanning domain of the Clostridium septicum alpha toxin. J Biol Chem 2004, 279:14315-14322. The membrane-penetrating region was identified from the model of AT based on the aerolysin structure. Fluorescence and mutagenesis were used to confirm that a loop forms the amphipathic b-hairpin, providing the first experimental data on the pore-forming region of the aerolysin family. 32. Petosa C, Collier RJ, Klimpel KR, Leppla SH, Liddington RC: Crystal structure of the anthrax toxin protective antigen. Nature 1997, 385:833-838. 33. Santelli E, Bankston LA, Leppla SH, Liddington RC: Crystal structure of a complex between anthrax toxin and its host cell receptor. Nature 2004, 430:905-908. 34. Lacy DB, Wigelsworth DJ, Melnyk RA, Harrison SC, Collier RJ: Structure of heptameric protective antigen bound to an anthrax toxin receptor: a role for receptor in pH-dependent pore formation. Proc Natl Acad Sci USA 2004, 101:13147-13151. The authors present structures of the anthrax PA prepore at 3.6 A˚ and the prepore–receptor complex at 4.3 A˚. This shows, for the first time, the position of the pore-forming loops that form the b-hairpin and also that the receptor binds to both domains 2 and 4, indicating that this interaction prevents premature formation of the pore. 35. Rossjohn J, Feil SC, McKinstry WJ, Tweten RK, Parker MW: Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form. Cell 1997, 89:685-692. 36. Polekhina G, Giddings KS, Tweten RK, Parker MW: Insights into the action of the superfamily of cholesterol-dependent cytolysins from studies of intermedilysin. Proc Natl Acad Sci USA 2005, 102:600-605. The second structure of a CDC, intermedilysin, confirms the highly conserved structure of this family, and indicates that the link between domain 4 and the remainder of the protein is flexible. Mutagenesis of the conserved tryptophan motif locks the toxin in the prepore state. 37. Solovyova AS, Nollmann M, Mitchell TJ, Byron O: The solution structure and oligomerization behavior of two bacterial toxins: pneumolysin and perfringolysin O. Biophys J 2004, 87:540-552. 38. Giddings KS, Zhao J, Sims PJ, Tweten RK: Human CD59 is a receptor for the cholesterol-dependent cytolysin intermedilysin. Nat Struct Mol Biol 2004, 11:1173-1178. 39. Shepard LA, Heuck AP, Hamman BD, Rossjohn J, Parker MW, Ryan KR, Johnson AE, Tweten RK: Identification of a membrane-spanning domain of the thiol-activated poreforming toxin Clostridium perfringens perfringolysin O: an a-helical to b-sheet transition identified by fluorescence spectroscopy. Biochemistry 1998, 37:14563-14574. Current Opinion in Structural Biology 2006, 16:230–236
236 Macromolecular assemblages
40. Shatursky O, Heuck AP, Shepard LA, Rossjohn J, Parker MW, Johnson AE, Tweten RK: The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins. Cell 1999, 99:293-299.
44. Bittencourt SE, Silva LP, Azevedo RB, Cunha RB, Lima CM, Ricart CA, Sousa MV: The plant cytolytic protein enterolobin assumes a dimeric structure in solution. FEBS Lett 2003, 549:47-51.
41. Tilley SJ, Orlova EV, Gilbert RJ, Andrew PW, Saibil HR: Structural basis of pore formation by the bacterial toxin pneumolysin. Cell 2005, 121:247-256. Fitting perfringolysin into cryo-EM maps of the prepore and pore states of pneumolysin shows that the toxin domains separate and that domain 2 collapses down onto the membrane, allowing insertion of the b-hairpins to form the pore wall.
45. Mancheno JM, Tateno H, Goldstein IJ, Martinez-Ripoll M, Hermoso JA: Structural analysis of the Laetiporus sulphureus hemolytic pore-forming lectin in complex with sugars. J Biol Chem 2005, 280:17251-17259.
42. Czajkowsky DM, Hotze EM, Shao Z, Tweten RK: Vertical collapse of a cytolysin prepore moves its transmembrane b-hairpins to the membrane. EMBO J 2004, 23:3206-3215. Atomic force microscopy measurements show that the prepore-to-pore conversion of perfringolysin is accompanied by a height reduction of 40 A˚. 43. Giddings KS, Johnson AE, Tweten RK: Redefining cholesterol’s role in the mechanism of the cholesterol-dependent cytolysins. Proc Natl Acad Sci USA 2003, 100:11315-11320.
Current Opinion in Structural Biology 2006, 16:230–236
46. Sher D, Fishman Y, Zhang M, Lebendiker M, Gaathon A, Mancheno JM, Zlotkin E: Hydralysins, a new category of beta-pore-forming toxins in cnidaria. J Biol Chem 2005, 280:22847-22855. 47. Lin SC, Lo YC, Lin JY, Liaw YC: Crystal structures and electron micrographs of fungal volvatoxin A2. J Mol Biol 2004, 343:477-491. 48. Tieleman DP, Leontiadou H, Mark AE, Marrink SJ: Simulation of pore formation in lipid bilayers by mechanical stress and electric fields. J Am Chem Soc 2003, 125:6382-6383.
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