Pore-forming toxins (PFT) are water-soluble proteins that possess the remarkable ability to self-assemble on the membrane of target cells, where they form pores causing cell damage. Here, we elucidate the mechanism of action of the haemolytic protein fragaceatoxin C (FraC), a α-barrel PFT, by determining the crystal structures of FraC at four different stages of the lytic mechanism, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore. The structure of the transmembrane pore exhibits a unique architecture composed of both protein and lipids, with some of the lipids lining the pore wall, acting as assembly cofactors. The pore also exhibits lateral fenestrations that expose the hydrophobic core of the membrane to the aqueous environment. The incorporation of lipids from the target membrane within the structure of the pore provides a membrane-specific trigger for the activation of a haemolytic toxin.

The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore-forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.

Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3-4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membraneembedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.Targeting and attachment of proteins to membranes is one of the key steps in many cellular processes (1-3). Protein-membrane interactions have been studied intensively in recent years with many different examples of proteins and membranes. These interactions can be promoted at the lipid-water interface by lipid anchors, electrostatic forces or surface-exposed aromatic and aliphatic residues (1, 2, 4). Compared with protein-protein interactions, details of protein-membrane interactions are poorly defined. Some of the best characterized examples are a phospholipase C pleckstrin homology domain specific for phosphatidylinositol trisphosphate (5) and small protein kinase-C-conserved (C2) domains specific for zwitterionic, particularly phosphatidylcholine membranes (6).Another group of proteins interacting with lipid membranes are pore-forming toxins (PFT) 1 (7-10), which bind to membranes before eliciting their toxic effects via the formation of transmembrane pores. The most studied PFT are bacterial since this group includes important virulence factors. Few examples of eukaryotic PFT have been well characterized, exceptions being the actinoporins, cytolysins found exclusively in sea anemones (10, 11). Members of this family have properties distinct from other PFT: they are composed of 175-179 amino acids, contain no cysteine residues, have pIϾ9.5, and show a preference for sphingomyelin (SM)-containing membranes. Actinoporins act on cellular and model lipid membranes by forming cation-selective pores with a hydrodynamic diameter of ϳ2 nm. The mechanism of pore formation involves at least two steps: binding of the water soluble m...

Equinatoxin-II is a eukaryotic pore-forming toxin belonging to the family of actinoporins. Its interaction with model membranes is largely modulated by the presence of sphingomyelin. We have used large unilamellar vesicles and lipid monolayers to gain further information about this interaction. The coexistence of gel and liquid-crystal lipid phases in sphingomyelin/phosphatidylcholine mixtures and the coexistence of liquid-ordered and liquiddisordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol mixtures favor membrane insertion of equinatoxin-II. Phosphatidylcholine vesicles are not permeabilized by equinatoxin-II. However, the localized accumulation of phospholipase C-generated diacylglycerol creates conditions for toxin activity. By using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces between coexisting lipid phases may function as preferential binding sites for the toxin. The possible implications of such a mechanism in the assembly of a toroidal pore are discussed.Equinatoxin II (Eqt-II) 1 is a member of the actinoporins, a group of sea anemone cytolysins (1). It is a 179-amino acid residue protein with a molecular mass of 19.8 kDa and an isoelectric point of 10.5 (2). Its three-dimensional structure has been solved by x-ray crystallography and NMR (3, 4). Eqt-II forms cation-selective pores with a diameter of ϳ2 nm in cell and model membranes (5-7). The mechanism of pore formation is a multistep process consisting of (i) membrane binding of the water-soluble monomer, (ii) oligomerization on the membrane surface, and (iii) pore formation (1,(5)(6)(7)(8)(9)(10)(11). This mechanism is common to other actinoporins like sticholysin-II from Stichodactyla helianthus (12, 13). Membrane insertion of Eqt-II and sticholysins is favored by the presence of sphingomyelin within the target membrane (6, 8, 14 -16). The recent finding of a phosphocholine binding site in the three-dimensional structure of sticholysin-II (13) supports the role of sphingomyelin as a specific receptor for actinoporins, as other authors have suggested (17, 18). However, the presence of sphingomyelin is not strictly necessary for the lytic activity of these toxins, which are also active in phosphatidylcholine/cholesterol mixtures (14, 16). Therefore, other factors are likely to govern their mechanism of action.Mixtures of sphingomyelin, phosphatidylcholine, and cholesterol are characteristic of the so-called rafts, microdomains in which the concentration of membrane components (lipids or proteins) and their physicochemical properties are different from the surrounding environment. The increasing amount of information pointing to the existence of lipid domains in cell and model membranes and their implication in many crucial biological processes has been extensively reviewed (19 -26). One important characteristic of rafts is their resistance to detergent solubilization (27)(28)(29)(30). This property is associated with the fact...

Pore-forming toxins (PFTs) are proteins that are secreted as soluble molecules and are inserted into membranes to form oligomeric transmembrane pores. In this paper, we report the crystal structure of Fragaceatoxin C (FraC), a PFT isolated from the sea anemone Actinia fragacea, at 1.8 Å resolution. It consists of a crown-shaped nonamer with an external diameter of about 11.0 nm and an internal diameter of approximately 5.0 nm. Cryoelectron microscopy studies of FraC in lipid bilayers reveal the pore structure that traverses the membrane. The shape and dimensions of the crystallographic oligomer are fully consistent with the membrane pore. The FraC structure provides insight into the interactions governing the assembly process and suggests the structural changes that allow for membrane insertion. We propose a nonameric pore model that spans the membrane by forming a lipid-free α-helical bundle pore.