Pore Formation: an Ancient Yet Complex Form of Attack ⁎ Ioan Iacovache, F

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Biochimica et Biophysica Acta 1778 (2008) 1611–1623 www.elsevier.com/locate/bbamem

Review Pore formation: An ancient yet complex form of attack ⁎ Ioan Iacovache, F. Gisou van der Goot , Lucile Pernot

Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Faculty of Life Sciences, Station 15, CH 1015 Lausanne, Switzerland Received 13 November 2007; received in revised form 3 January 2008; accepted 4 January 2008 Available online 12 February 2008

Abstract

Bacteria, as well as higher organisms such as sea anemones or earthworms, have developed sophisticated virulence factors such as the pore- forming toxins (PFTs) to mount their attack against the host. One of the most fascinating aspects of PFTs is that they can adopt a water-soluble form at the beginning of their lifetime and become an integral transmembrane protein in the membrane of the target cells. There is a growing understanding of the sequence of events and the various conformational changes undergone by these toxins in order to bind to the host cell surface, to penetrate the cell membranes and to achieve pore formation. These points will be addressed in this review. © 2008 Elsevier B.V. All rights reserved.

Keywords: Pore-forming toxin; Perforin; Cholesterol-dependent cytolysin; Aerolysin; Actinoporin

Contents

1. Introduction...... 1611 2. Structural classification of pore-forming toxins...... 1612 2.1. α-PFT ...... 1612 2.1.1. Colicins...... 1612 2.1.2. Actinoporins ...... 1612 2.1.3. Insecticidal pore-forming toxins of the Cry family...... 1615 2.2. The ß-PFTs ...... 1615 2.2.1. S. aureus PFTs...... 1615 2.2.2. Aerolysin and related toxins ...... 1616 2.2.3. The protective antigen of Anthrax toxin ...... 1616 2.2.4. Cholesterol-dependent cytolysins (CDCs) ...... 1618 3. Membrane insertion of PFTs ...... 1619 3.1. Membrane insertion of α-PFTs ...... 1619 3.2. Membrane insertion of ß-PFTs ...... 1619 4. Conclusion and future perspectives ...... 1619 Acknowledgements ...... 1620 References ...... 1620

1. Introduction well as sea anemones or earthworms, produce toxic substances able to disrupt cellular membranes [1–3]. These can be small Whether it is a defense mechanism or a means to attack the chemical compounds (cyclodextrin family [4], lipids etc), pep- host at the onset of infection, many pathogenic organisms as tides or large protein assemblies, all produced with the common aim of altering the lipid bilayer of target cells. ⁎ Corresponding author. Tel.: +41 21 6931791; fax: +41 21 6939538. The plasma membrane of a cell is not just a simple physical E-mail address: [email protected] (F.G. van der Goot). barrier that separates the cytoplasm from the outer world but

0005-2736/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2008.01.026 1612 I. Iacovache et al. / Biochimica et Biophysica Acta 1778 (2008) 1611–1623 constitutes one of the most complex components of the cell. The [22–24], actinoporins that are produced by sea anemones [19], picture of a membrane composed of a homogeneous lipid mixture diphtheria toxin produced by Corynebacterium diphtheriae,exo- in which proteins are embedded is indeed no longer valid [5,6].It toxin A from Pseudomonas aeruginosa [1] and the insecticidal has become increasingly apparent that the 2-dimensional com- Cry toxins produced by Bacillus thuringiensis [20,21] ,which partmentalization is an important requirement to fulfill the mul- received much attention due to their potential role in pest control tiple functions of the plasma membrane. Proteins cluster into in transgenic crops. homo or hetero assemblies within the membrane, lipids undergo lateral segregation and finally preferential assemblies of lipids 2.1.1. Colicins with proteins form. Being highly opportunistic in nature, bacterial Colicins are PFTs produced by E. coli and aimed at killing pathogens have learned to use this plasma membrane organization related E. coli [22]. The bacteria expressing the toxin also produce and in some instances perturb it [7–9]. an immunity protein that protects them from the action of the Among the toxins produced by bacteria, the largest class is toxin [22–24]. After production, the soluble colicins are secreted that of pore-forming toxins (PFTs), which represent some 30% in the external medium and upon encounter of the target cells bind of all known bacterial toxins [1] and will be the topic of this to specific receptors [25–27] and are subsequently translocated review. PFTs are however also produced by higher organisms into the periplasm via a variety of mechanisms [13,25,28].Once like cnidaria, [10], sea anemones (actinoporins) [11], earthworm in the periplasmic compartment, some colicins, such as Colicin A (lysenin) [2] and plants (enterolobin) [12]. Within the PFT or E1, form pores in the cytoplasmic membrane while others are family, some sub-families such as that of the cholesterol- translocated into the cytoplasm where they function as DNAse, dependent cytolysins (CDC) or the actinoporins can be found. RNAse or block protein synthesis [22,23]. It has been shown Although sequence similarities can be found within these fa- recently that endonuclease domains of colicins may interact with milies, PFTs from different sub-families share no sequence the bacterial cytoplasmic membrane and mediate their own homology, despite similarities in the mode of action. One of the translocation through the bacterial inner membrane [29,30].The most fascinating aspects of these toxins is that they are secreted structures of various pore-forming colicins have been solved to as fully soluble proteins yet in their final state, they form a atomic resolution [31–33] and reveal the multi-domain structure transmembrane channel in the target cell membrane. This is of these proteins. As an example, colicin Ia [32] can be divided achieved by the following common steps: the soluble toxin into three domains [34]: the translocation domain is formed by 3 diffuses toward the target cell to which it binds via specific helices, one of which extends to the receptor-binding domain. receptors. As will be described below, many PFTs then require This receptor-binding domain is formed by a short ß-hairpin and an oligomerization step that is followed by membrane insertion three short helices. Both domains, and the channel-forming and channel formation [13]. Interesting new findings have been domain, are connected by the coiled–coil structure (Fig. 1a). The made regarding the effects of pore formation in target cells, how coil–coiled domain is thought to be involved in bridging the the cell senses the effects of the toxins and subsequently react, periplasmic space based on the unusual length of the domain sometimes leading to membrane repair and cell survival. These ~160 Å. The pore-forming domain, made up of a bundle of ten α- topics will not be covered here and the interested reader is helices is a globular domain with a hydrophobic core composed of referred to the following recent reviews [14–16]. an α-helical hairpin (helices 8 and 9)(Fig. 1b). This α-helical bundle structure with a central hidden hydrophobic helical hairpin 2. Structural classification of pore-forming toxins is common to all pore-forming colicins and is also shared by completely unrelated but pore-forming proteins, such as the Pore-forming toxins can be classified in more than one-way, translocation domain of diphtheria toxin [35]. Unmasking of the for example according to their pore size or membrane-binding hydrophobic central hairpin, through partial unfolding of the mode. We prefer a classification according to the type of struc- pore-forming domain is thought to be triggered by a local low pH ture with which the toxin finally crosses the membrane, i.e. alpha at the membrane interface [13,36] although other mechanisms helical (α-PFT) or ß-barrel (ß-PFT) [17,18]. Interestingly one cannot be excluded. Channel formation however requires inser- reaches the same classification when sorting PFTs based on the tion not only of the central hydrophobic hairpin but also that of presence or absence of hydrophobic stretches in their primary adjacent helices [23,37,38]. Also multimerization is most likely sequence. α-PFTs indeed contain stretches of hydrophobicity required although definite proof is lacking. Unfortunately no that are predicted to be helical and as we will see are able to high-resolution structure of the pore configuration is available and span lipid bilayers. In contrast, ß-PFTs are not predicted to be therefore the exact configuration of the helices remains quite transmembrane based on the analysis of hydrophobicity. They speculative. however contain pairs of amphipathic ß-strands, which when combined in multi-protein structures generate a sufficiently hy- 2.1.2. Actinoporins drophobic surface for membrane insertion to occur. Actinoporins belong to the family of pore-forming toxins produced by sea anemones [11] that have received increasing 2.1. α-PFT attention in recent years [3]. The best-studied members of this family are equinatoxin II produced by Actinia equinia and The α-PFT family includes the colicins produced by Escher- sticholysin II of Stichodactyla helianthus. Binding of these toxins ichia coli, which are arguably the best-characterized in this family to membranes appears to be mediated by lipids [39,40]. I. Iacovache et al. / Biochimica et Biophysica Acta 1778 (2008) 1611–1623 1613

Fig. 1. Ribbon representation of the crystal structures of α-PFT; the transmembrane region is colored in violet (a) colicin Ia (PDB entry code 1CII): the T translocation, R receptor-binding domain and the C channel-forming domains are indicated (b) close-up view of the C pore-forming domain of colicin Ia (c) Equinatoxin II (PDB entry code 1IAZ) (d) Cry4Aa composed of domains I, II and III (PDB entry code 2C9K) (e) Domain I, the pore-forming domain of Cry4Aa. Figures were produced with the program PyMol (http://www.pymol.sourceforge.net). 1614 I. Iacovache et al. / Biochimica et Biophysica Acta 1778 (2008) 1611–1623 I. Iacovache et al. / Biochimica et Biophysica Acta 1778 (2008) 1611–1623 1615

Actinoporins are small proteins of ~20 kDa and their structure 2.2. The ß-PFTs reveals a β-sandwich flanked on both faces by short α-helices [39] (as shown for equinatoxin in Fig. 1c). The N-terminal ~30 The majority of bacterial PFTs known to date belong to residues that include one of the α-helices are free to detach from the ß-PFT class. Members include the large family of the the core of the protein and insert into the bilayer to form the pore. cholesterol-dependent cytolysins (CDCs) [53], the multiple Upon insertion the N-terminal helix is extended by 8–9residues PFTs produced by Staphylococcus aureus such as its α-toxin [41]. In order to form functional pores in the membrane, these [54], Bacillus thuringiensis Cyt delta-endotoxins [21] as well as toxins must oligomerize forming ring shaped tetramers. Each the translocation subunits of certain AB type toxins including monomer contributes one α-helix to the formation of a 2 nm wide that of anthrax toxin [55]. The means by which ß-PFT form pore [19]. transmembrane pores is better understood due to the availability of structures, not only of the soluble toxin but also of the 2.1.3. Insecticidal pore-forming toxins of the Cry family transmembrane form solubilized in detergents [56–58]. As for This family of α-pore-forming toxins has received increasing α-PFTs, ß-PFTs are secreted by bacteria, or other organisms, as attention due to their use as insecticides especially in the trans- soluble proteins that diffuse in the external medium towards genic crop [42]. As a general mode of action these toxins are target cells to which they bind via specific receptors. Once produced during sporulation of the bacterium as inclusion bound, ß-PFTs must oligomerize, a process favored by the crystals. Upon ingestion by the insect larvae and under alkaline concentration effect related to the change in dimension between conditions in the insect midgut, the crystals dissolve releasing the extracellular space (3D) and the membrane surface (2D). the monomeric pro-toxins [42]. Cry toxins have been found to Some members of the ß-PFT family require activation by host- bind to the GPI-anchored insect aminopeptidase N [43] and to specific proteases in order to be able to oligomerize [59,60]. The glycolipids in worms [44]. A sequential binding scenario has formed oligomer is for most toxins initially not transmembrane also been proposed where the toxin binds to a first receptor for and thus called the prepore [53]. A second series of confor- activation followed by an interaction with a secondary receptor mational changes is required for membrane insertion and pore that relocates the toxin to lipid microdomains that could favor formation to occur [13]. To date the only high-resolution struc- multimerization [42,45]. Recently however an interesting syn- ture of a oligomeric pore is that of the Staphylococcal α-toxin ergistic mechanism was proposed where the B. thuringiensis heptamer, the structure of which was solved in detergents [56]. Cyt1Aa toxin first bound to the target cell membrane and became the receptor for the Cry11Aa toxin [46]. Although it is 2.2.1. S. aureus PFTs clear that Cry toxins require multimerization, the stochiometry S. aureus produces a variety of PFTs, including α-toxin of the complex is a matter of debate, dimers, trimers, tetramers and the leukocidins, which as a family are arguably the best- and even large multimeric assemblies having been proposed characterized PFTs from a structural point of view, since high- (reviewed in [47]). Recent 2D crystallization trials showing a resolution structures are available for the soluble and oligomeric trimeric pore in either a propeller or pinwheel form and which forms. The leukocidins are bi-component toxins, pore formation were interpreted as closed/opened states of the channel seem to requiring both a so-called class F component and a class S tilt the balance in favor of the trimeric pore assembly [47]. component [61,62], whereas α-toxin forms homomeric pores The structure of several monomeric active Cry toxins has [56]. The structures of two components of the class F proteins, been solved [20,48–51] showing a large protein that, based also HlgB from γ-hemolysin [63] and LukF-PV from Panton– on biochemical evidence, can be divided into three separated Valentine leukocidin [64] as well as one S component, LukS-PV domains (Fig. 1d). The N-terminal, pore-forming, domain I is a [65] have been solved (Fig. 2a). To date no structural infor- helical globular domain in which helix 5 is strongly hydrophobic mation of a leukotoxin hetero-oligomer, whose proposed sto- and forms the core of the domain while helices 1–4 and 6–7 form chiometry varies from 6 to 8 [66–70], is available. In contrast, the outside wall shielding the hydrophobic helix from the whereas the structure of the soluble form is not available for aqueous media [20], a structure reminiscent of that of the pore- Staphylococcal α-toxin, that of the heptameric transmembrane forming domain of colicins (Fig. 1e). Domain II of the protein is configuration, solubilized in detergent, is. Since all Staphylo- a lectin-like domain composed mainly of β-sheets and is coccal PFTs are highly homologous, it is believed that the probably involved in the binding of the protein to the receptor. combined information from the various structures provides an The third domain of the protein is composed of a twisted β- overall view of the changes that lead to pore formation. We will sandwich and its role in the activity of the toxin is not well first describe the channel structure and subsequently compare it understood. It is thought to play a role in the stability of the toxin to that of the soluble toxin. and to protect the main body of the protein from proteolytic All attempts to find a proteinaceous or glycolipid receptor activity and it might also have a role in receptor recognition and for α-toxin have failed. It has been recently proposed that high binding [48,52]. affinity binding of the toxin to the membrane could be mediated

Fig. 2. Ribbon representation of the crystal structures of ß-PFT: the transmembrane region is colored in purple (a) the water-soluble LukS-PV monomer with the pre- stem domain (PBD entry code 1T5R) (b) one α-toxin monomer in the oligomer; noted are the cap with the amino-latch, the rim and the stem domains (PDB entry code

7AHL) (c) proaerolysin (PDB entry code 1PRE) (d) anthrax PA: one monomer from PA63 heptameric prepore is represented (PDB entry code 1TZO) (e) perfringolysin O, where the conserved undecapeptide is colored in blue (PDB entry code 1PFO). Figures were produced with the program PyMol (http://www.pymol.sourceforge.net). 1616 I. Iacovache et al. / Biochimica et Biophysica Acta 1778 (2008) 1611–1623 by clusters of phosphocholine head groups of sphingomyelin, doproteases [89], which remove some 40 amino-acids from the within sphingomyelin cholesterol microdomains [7].Once C-terminus [90]. If the concentration is high enough, aerolysin ontheplasmamembrane,α-toxin (also called α-hemolysin) will subsequently oligomerize into a heptameric ring [91,92]. oligomerizes into a circular, water-soluble, heptameric prepore The X-ray structure of proaerolysin in the dimeric form revealed [63,71,72]. A second conformational change leads to mem- an L-shaped elongated molecule divided into two domains, a brane penetration of a fairly small portion of each monomer and small globular domain, and a large lobe linked together by a long formation of a water-filled transmembrane pore. The struc- stretch of residues [93] (Fig. 2c). The globular domain, domain ture of the detergent-solubilized α-toxin heptamer was solved 1, is mainly implicated in binding of the toxin to sugar mo- by X-ray crystallography, with a 1.9 Å resolution [56] (Fig. 3a). difications of the target receptor [86]. The elongated domain of The pore is a mushroom-shaped, hollow heptamer (with overall the protein has been traditionally divided into three distinct dimensions of 100 Å×100 Å), mostly composed of ß-structure domains that are non-continuous in sequence: domain 2 is and containing 3 domains (Fig. 2bandFig. 3a). The cap domain involved in binding to the GPI-anchor [84] and in initiating the consists of the ß-sandwich and an amino-latch that provides oligomerization process [94]. Domain 3 is responsible to some additional links between the protomers. Underneath the cap, degree for monomer–monomer contact in the heptamer [95]. the rim domain is located in close proximity of the outer leaflet Also it contains a loop shielded against the ß-sheet of the main of the plasma membrane. The stem domain, or the transmem- body that must move for oligomerization to proceed [96]. Re- brane ß-barrel pore perforating the membrane, consists of a 14- cently, the role of this loop was reinvestigated and it is actually stranded ß-barrel to which each protomer contributes an anti- the part of the protein that inserts into the membrane to form the parallel ß-hairpin. The diameter of the pore varies along its transmembrane amphipathic β-barrel [97,98] Finally, domain 4 lumen from 30 Å at the entrance to 22 Å along the stem around contains the pro-peptide, which is proteolytically removed upon [54]. Although several studies, using crystallography or mass activation [99]. The role of this pro-peptide might be to prevent spectrometry, reported the assembly of a heptameric pore, ato- oligomerization as shown for Clostridial alpha-toxin [100]. mic force microscopy experiments suggested that α-toxin No high-resolution structure is available of aerolysin in the could also associates into a hexameric pore [73]. transmembrane state. Cryo-negative staining EM analysis was The structures of the soluble PFTs, HlgB, LukF and LukF-PV however performed on the heptameric form of an aerolysin [63–65] are all very similar to that of the α-toxin protomer mutant [57]. A single point mutation Y221G was found to (Fig. 2a). The rim domains are rotated slightly with respect to the convert the wild-type aerolysin hydrophobic heptamer into cap domain. The main difference is as expected in the stem water-soluble complex. The molecular envelope obtained at region: in the water-soluble state, this region adopts a 3-stranded 13 Å revealed a conserved mushroom-shaped soluble heptamer amphipathic β-sheet, where the hydrophobic residues (which (Fig. 3b), resembling the previously observed pores of wild- face the lipids in the transmembrane configuration) pack against type aerolysin obtained by 2D crystallography [91] Docking of the β-sandwich. Another difference between the water-soluble the model of the wild-type proaerolysin where the C-terminal and membrane forms occurs in the 12 amino terminal residues of pro-peptide was removed allowed to propose a refined model the protein, the so-called N-terminal latch, which form a strand for the wild-type heptamer. Although location of domains 1 and in the soluble form but displays aperiodic structure in the 2 in cap of the mushroom was consistent with all experimental channel form. data, position of domain 3 and 4 in the mouth and lumen of the wild-type pore does not corroborate the finding that only a small 2.2.2. Aerolysin and related toxins loop in domain 3 is involved in spanning the membrane [97]. Aerolysin is a ß-PFT produced by Aeromonas hydrophila. Understanding pore formation by members of the aerolysin Related toxins, in terms of sequence and structure, are however family will therefore require further structural analysis of the produced by a great variety of organisms: α-toxin from Clos- channel form. tridium septicum [74], ε-toxin by Clostridium perfringens [75], Interestingly, as mentioned above, a novel class of pore- hydralysins by Cnidaria [10], a hemolytic lectin by the parasitic forming toxins has been recently discovered in the cnidaria mushroom Laetiporus sulphureus [76] and enterolobin by the phylum that belongs to the ß-pore formers and shows a re- seeds of the Brazilian tree Enterolobium contorliquum [77,78]. markable structural similarity to aerolysin [10]. The best-characterized member however remains aerolysin, which will thus be described in more detail. 2.2.3. The protective antigen of Anthrax toxin A. hydrophila secretes a precursor toxin, proaerolysin, via a Anthrax toxin, produced by Bacillus anthracis, the causative type II secretion system in the extracellular medium [79] and for agent of anthrax, belongs to the family of AB toxins (for a recent a recent review see [80]. Proaerolysin binds to the target mem- review see [55]). This ternary toxin consist of two enzymatic A brane, in a monomeric or dimeric form [81,82], via the specific moieties, Lethal factor (LF) and Edema Factor (EF), and one B interaction with glycosyl phosphatidyl inositol (GPI)-anchored moiety, the Protective Antigen (PA), which binds to the receptors proteins [83–86]. The glycan core and the N-linked sugar and translocates the A subunits across the membrane into the located on the proteinaceous moiety of the receptors are the cytoplasm. LF is a Zn2+ protease that cleaves and inactivates two determinants involved in the binding [86,87]. Proaerolysin MAP kinase kinases and EF a Ca2+- and calmodulin-dependent is converted into aerolysin by gut proteases, Aeromonas pro- adenylate cyclase [55]. PA, which is of interest to the topic of this teases [88] or members of the furin family of mammalian en- review, binds to one of the two identified receptors, TEM8, I. Iacovache et al. / Biochimica et Biophysica Acta 1778 (2008) 1611–1623 1617

Fig. 3. Ribbon representation of the crystal structures of β-PFT in their transmembrane form; the transmembrane region is colored in violet (a) α-toxin heptamer (PDB entry code 7AHL) (b) Molecular envelope of aerolysin Y221G heptamer. The image shows a dimer of heptamers joined together by their large base (reproduced with permission) (c) Prepore PA63 of the anthrax protective antigen (PDB entry code 1TZO) (d) Molecular envelope of the pneumolysin prepore and pore (reproduced with permission). Figures were produced with the program PyMol (http://www.pymol.sourceforge.net), except (b) and (d). 1618 I. Iacovache et al. / Biochimica et Biophysica Acta 1778 (2008) 1611–1623 tumor endothelial marker-8 [101], and GMG2, capillary mor- LLO [114] and Streptococcus intermedius Intermedilysin ILY phogenesis protein 2 [102]. As aerolysin, PA requires proteolytic [8]. ILY was found to bind to human CD59 [8], which is a GPI- activation. A fragment of 20 kDa is removed from the N-terminus anchored molecule required to prevent the formation of the and the remaining protein named PA63 stays bound to the receptor complement membrane attack complex (MAC) on cells by and undergoes circularly assembly into a heptameric prepore [55]. binding complement proteins C8a and C9 [115,116]. Unlike The PA prepore acts as the receptor for LF and/or EF [55].The aerolysin that binds to sugars on GPI-anchored proteins, ILY complex is then internalized and delivered to endosomes. The binds to the protein itself and more specifically to the same site prepore undergoes a pH dependent conformational change that on CD59 where binding of C8a and C9 occur [8]. However, even leads to membrane insertion. LF and EF unfurl and translocate in the case of ILY, cholesterol is required for pore formation trough the pore to the cytosolic compartment where they reach [117]. Possibly due to this requirement for cholesterol, CDCs and modify their cytosolic targets [55]. were shown to associate with cholesterol-rich lipid microdo- The X-ray structure of the monomeric PA [103] as well as that mains [118,119] and even lead to their aggregation [120]. of PA bound to its CMG2 receptor [104] displays an elongated The most interesting structural aspect of CDCs is that they molecule divided in 4 domains (Fig. 2d). Domain 1 contains the form very large pore of ≈300 Å in diameter and are composed cleavage site for processing PA83 into an activated PA63. Domain of up to 50 monomers, the number being somewhat variable 2 contains a large flexible loop named 2ß2–2ß3 that corresponds [121] as opposed to the above described ß-PFTs. CDCs not only to the “to be” amphipathic transmembrane ß-hairpin [105,106]. form oligomers with more protomers than other ß-PFTs but in Domain 3 is thought to participate in the oligomerization step. addition, each monomer contributes 2 ß-hairpins to the trans- Domain 4 is mainly involved in the interaction with the receptor. membrane ß-barrel [121,122], as opposed to one per monomer The structure of the PA63 heptamer has been solved on its own as for other ß-PFTs, thus leading to ß-barrels composed of some well as bound to the PA binding domains of the receptors 200 strands [58]! [103,107]. These structures show that the 2ß2–2ß3 loop projects Two structures of the soluble forms of CDCs are currently out of one monomer and inserts into the neighboring monomer available, that of PFO [123] and that of ILY [124].Asex- between domains 2 and 4 (Fig. 3c), which both interact with the pected from the sequence similarity, they share a similar glo- receptor. Therefore, for channel formation to occur, separation of bal architecture: an elongated molecule composed primarily of domains 2 and 4 must take place to free this loop. The interface β-sheets and divided in 4 domains (Fig. 2e). As in the case of between domains 2 and 4 contains seven histidine residues that proaerolysin, at a first glance, one can differentiate an elongated upon acidification in the endosome luminal will be protonated domain (composed by domains 1, 2 and 4) and a separated leading to the repulsion between the two domains and liberation domain (domain 3) that is made up by a β-sheet and several short of the pore-forming loop. It is interesting to note that cellular α-helices. Domain 4 is involved in binding to cholesterol-rich receptors do more than just binding PA to the cells surface. It was membrane domains through the insertion of several loops: a shown that the receptor determines the pH at which the pore- highly conserved undecapeptide rich in tryptophan residues and 3 forming loop will unfold [108,109] and may also serve to orient hydrophobic short loops called L1, L2 and L3 [125–127].The PA properly with respect to the membrane [110]. The structure of interactions of PFO with cholesterol are crucial since they prime the transmembrane form has not been solved. A model has irreversible conformational changes that are propagated along the however been proposed showing a very long ß-barrel of which toxin and are important for the prepore to pore conversion [113]. only the very end would perforate the membrane, the rest would Crystallization of PFO at two different pHs showed that con- bridge the length between the PA binding domain of the receptor formational changes in domain 3 are connected with those oc- and the membrane surface [104]. curring in domain 4 [128]. Domain 3 contains the two segments that will ultimately form 2.2.4. Cholesterol-dependent cytolysins (CDCs) the two transmembrane β-hairpins (TMH1 and TMH2) (Fig. 2e) CDCs compose a large family of more than 20 members [121,122]. TMH1 and TMH2 are interestingly in an α-helical [53]. These major virulence factors are secreted by a variety of configuration in the soluble form, thus undergoing a prion like Gram-positive bacteria including perfringolysin O (PFO) from alpha to beta transition upon pore formation [129]. Pores of C. perfringens, streptolysin O (SLO) from Streptococcus pyo- CDCs have at present not been crystallized. Electron microscopy genes, pneumolysin from S. pneumoniae and listeriolysin O studies have however provided a very clear picture of these (LLO) from Listeria monocytogenes.CDCsshareahighde- beautifully arranged complexes [58] and suggest conformational gree of sequence similarity (40–80%) suggesting that they have changes that accompany membrane insertion (Fig. 3d). similar structures and activity [111]. Pore-forming proteins are not unique to the bacterial world. As the name indicates, CDCs require cholesterol for their The mammalian immune systems utilizes pore formation as a action. The exact step at which cholesterol is required – cell mean to kill pathogens or infected cells through the action of the surface binding, oligomerization or membrane insertion – varies complement cascade, which final steps are the formation of the between CDCs [112]. It was initially thought that cholesterol membrane attack complex (MAC), or perforin secreted by cyto- was the receptor for all CDCs. PFO indeed directly binds to lytic T cells. These proteins share a common MACPF domain, cholesterol and even in solution this interaction leads to con- which was recently solved for two proteins: Plu–MACPF from formational changes throughout the protein that promote mul- Photorhabdus luminescens [130] and the human C8a [131]. timerization [113]. Cholesterol is however not the receptor for Remarkably, the MACPF domain was found to be structurally I. Iacovache et al. / Biochimica et Biophysica Acta 1778 (2008) 1611–1623 1619 homologous to CDCs, also harboring TMH1 and TMH2 do- be driven by an initial insertion of hydrophobic helices in the mains, indicating that bacterial and mammalian membranolytic case of ß-PFTs, insertion has to be a concerted event. It has been proteins share a common mechanism for membrane insertion. shown for the protective antigen of anthrax toxin that specific residues are required to position the ß-hairpin in the good re- 3. Membrane insertion of PFTs gister through salt bridges [141]. From a sequence analysis of known or predicted transmem- Very little is known about this crucial step of the mode of brane regions in ß-PFTs based on the residues that line the pore action of PFTs. As mentioned, α-PFTs contain hydrophobic and form the tip of the anti-parallel transmembrane ß-hairpin, a helices, the membrane insertion of which, when exposed, is model has been proposed that would divide the pores into two thought to be spontaneous. In the case of ß-PFTs, oligomeriza- categories [97]. In the case of α-toxin of S. aureus, the pore is tion always precedes membrane insertion. What triggers the lined with short polar residues lacking any charge while the tip prepore to pore conversion however remains largely obscure. of the hairpin contains three charged residues. It is possible that Prepore formation, while described in many ß-PFTs such as the insertion proceeds due to the hydrophobicity of the residues CDCs [53], S. aureus α-toxin [132] or C. septicum α-toxin that will form the external face of the pore in the final structure [100], was however never observed for aerolysin and it appears while the charged tip is hidden towards the inside of the channel that heptamer formation and membrane insertion are concomi- during membrane penetration. Once on the other side of the tant events [97]. We have indeed proposed that ß-barrel folding membrane, the charges will anchor the pore in the inserted and membrane insertion are coupled as also proposed for the conformation. In the case of aerolysin, the inside of the pore is folding of outer membrane proteins [133]. lined with charged residues except for the tip of the hairpins, which are hydrophobic [97]. In this case, insertion would be 3.1. Membrane insertion of α-PFTs initiated by the tips of the ß-hairpins, which, once on the other side, would fold back towards the hydrophobic core of the As mentioned earlier, the pore-forming domain of colicins membrane anchoring the pore in a rivet like manner [97]. contains a hydrophobic helical hairpin formed by helices 8 and 9. The composition of the plasma membrane could also play a These are initially hidden within the helical bundle but become role in promoting membrane insertion, either by promoting the exposed upon partial unfolding of the molecule. Membrane oligomerization process as do lipid rafts [9,119,142],orby insertion of these helices leads to the formation of a pore that is promoting the formation of inverted phases [143]. It has indeed at that stage still closed, the so-called umbrella model [23].In been shown using artificial liposomes that membrane insertion the next step of channel formation, the rest of the helices of the of aerolysin was favored by lipids such as phosphatidyletha- C-domain are thought to insert into the bilayer leading to the open nolamine and diacylglycerol [143]. The underlying mechanism pore [134]. It is not clear how a monomeric toxin can form a pore however remains to be unraveled. and it has thus been proposed that colicin needs to multimerize for pore formation to occur. Recently however, it has been proposed 4. Conclusion and future perspectives that the colicin pore is also lined with lipids forming a toroidal pore complex [135] such as the pores formed by small peptides or Pore-forming toxins have long fascinated scientists both the translocation channel form by diphtheria toxin [136].Recent through their structural properties and mode of action [144, evidence points to this toroidal pore complex as a general 145]. Many cellular processes and defense mechanisms were mechanism even in the case of oligomeric toxins assemblies such unraveled by studying the opportunistic behavior of pathogens as the pores made by actinoporins [137]. In this latter case, the whose toxins have evolved to hijack specific cellular processes lipids would play a major role in pore formation and indeed it has and whose action trigger specific cellular responses [14,146]. been shown that certain lipids favor pore formation while other From this point of view it is clear that the study of pore-forming can inhibit the toxin activity, a property that was related with the toxins, from bacteria or other organisms, would not only offer capacity of the lipids to favor either positive or negative curvature ways to counter attacks but answer more general questions from [138,139]. While a toroidal pore formed by small pore-forming a broad spectra of biology. peptide is not a stable assembly due to the lack of a rigid structure The newly described structure of Plu–MACPF from Pho- connecting the α-helices that span the membrane, in the case of torhabdus luminescens [130] andthehumanC8a[131] again PFTs, the pores are more stable [13].Thisismostprobablyduein shows that knowledge gained in the pore-forming toxins field part to the fact that these helices are kept together by extracellular can be broadened and applied to defense mechanism of higher interactions. Stability can also be increased by anchoring pores to organisms. Indeed, as mentioned before, pore-forming toxins the trans-side of the membrane, a mechanism proposed both for are ancient weapons that are not exclusively bacterial but also equinatoxin [140] and aerolysin [97] and can also be induced by foundinplants[78],cnidaria[3,10] and mammals [130,131]. designing mutants of the transmembrane helices [36]. 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