Eukaryotic Pore-forming Toxin Research Articles

Neutron reflection study of the interaction of the eukaryotic pore-forming actinoporin equinatoxin II with lipid membranes reveals intermediate states in pore formation

Equinatoxin II (EqtII), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal α-helix into the membrane. EqtII pore formation is dependent on sphingomyelin (SM), although cholesterol (Chol) and membrane microdomains have also been suggested to enhance its activity. We have investigated the mechanism of EqtII binding and insertion by using neutron reflection to determine the structures of EqtII–membrane assemblies in situ. EqtII has several different modes of binding to membranes depending on the lipid composition. In pure dimyristoyl-phosphatidylcholine (DMPC) membranes, EqtII interacts weakly and reversibly with the lipid head groups in an orientation approximately parallel to the membrane surface. The presence of sphingomyelin (SM) gives rise to a more upright orientation of EqtII, but Chol is required for insertion into the core of the membrane. Cooling the EqtII–lipid assembly below the lipid phase transition temperature leads to deep water penetration and a significant reduction in the extension of the protein outside the membrane, indicating that phase-separation plays a role in EqtII pore-formation. An inactive double-cysteine mutant of EqtII in which the α-helix is covalently tethered to the rest of the protein, interacts only reversibly with all the membranes. Releasing the α-helix in situ by reduction of the disulphide bridge, however, causes the mutant protein to penetrate in DMPC–SM–Chol membranes in a manner identical to that of the wild-type protein. Our results help clarify the early steps in pore formation by EqtII and highlight the valuable information on protein–membrane interactions available from neutron reflection measurements.

Cys mutants in functional regions of Sticholysin I clarify the participation of these residues in pore formation

Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg52 in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu2, Phe15) did not modify the toxin’s permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation.

Selective permeabilization of the host cell membrane of Plasmodium falciparum-infected red blood cells with streptolysin O and equinatoxin II

Plasmodium falciparum develops within the mature RBCs (red blood cells) of its human host in a PV (parasitophorous vacuole) that separates the host cell cytoplasm from the parasite surface. The pore-forming toxin, SLO (streptolysin O), binds to cholesterol-containing membranes and can be used to selectively permeabilize the host cell membrane while leaving the PV membrane intact. We found that in mixtures of infected and uninfected RBCs, SLO preferentially lyses uninfected RBCs rather than infected RBCs, presumably because of differences in cholesterol content of the limiting membrane. This provides a means of generating pure preparations of viable ring stage infected RBCs. As an alternative permeabilizing agent we have characterized EqtII (equinatoxin II), a eukaryotic pore-forming toxin that binds preferentially to sphingomyelin-containing membranes. EqtII lyses the limiting membrane of infected and uninfected RBCs with similar efficiency but does not disrupt the PV membrane. It generates pores of up to 100 nm, which allow entry of antibodies for immunofluorescence and immunogold labelling. The present study provides novel tools for the analysis of this important human pathogen and highlights differences between Plasmodium-infected and uninfected RBCs.

The equinatoxin N‐terminus is transferred across planar lipid membranes and helps to stabilize the transmembrane pore

Equinatoxin II is a cytolytic protein isolated from the sea anemone Actinia equina. It is a member of the actinoporins, a family of eukaryotic pore-forming toxins with a unique mechanism of pore formation. Equinatoxin II is a 20 kDa cysteineless protein, with sphingomyelin-dependent activity. Recent studies showed that the N-terminal region of the molecule requires conformational flexibility during pore formation. An understanding of the N-terminal position in the final pore and its role in membrane insertion and pore stability is essential to define the precise molecular mechanism of pore formation. The formation of pores and their electrophysiologic characteristics were studied with planar lipid membranes. We show that amino acids at positions 1 and 3 of equinatoxin II are exposed to the lumen of the pore. Moreover, sulfhydryl reagents and a hexa-histidine tag attached to the N-terminus revealed that the N-terminus of the toxin extends through the pore to the other (trans) side of the membrane and that negatively charged residues inside the pore are crucial to define the electrophysiologic characteristics of the channel. Finally, we detected a new, less stable, state with a lower conductance by using a deletion mutant in which the first five N-terminal amino acids were removed. We propose that the first five amino acids help to anchor the amphipathic helix on the trans side of the membrane and consequently stabilize the final transmembrane pore.

Membrane binding of zebrafish actinoporin-like protein: AF domains, a novel superfamily of cell membrane binding domains.

Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomsen-Friedenreich antigen. In the present study we found a number of sequences in public databases with similarity to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the membrane binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in Escherichia coli. It displays membrane-binding behaviour but does not have permeabilizing activity or sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit-body lectin family comprise a novel superfamily of membrane binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).

Properties of nonfused liposomes immobilized on an L1 Biacore chip and their permeabilization by a eukaryotic pore-forming toxin

The L1 chip is used intensively for protein–membrane interaction studies in Biacore surface plasmon resonance systems. The exact form of captured lipid membranes on the chip is, however, not precisely known. Evidence exists that the vesicles both remain intact after the binding to the chip and fuse to form a large single-bilayer membrane. In this study, we were able to bind up to approximately 11,500 resonance units of zwitterionic liposomes (100 nm in diameter) at a low flow rate. We show by fluorescence microscopy that the entire surface of the flow cell is covered homogeneously by liposomes. Negatively charged vesicles (i.e., those composed of phosphatidylcholine/phosphatidylglycerol [1:1]) always deposited less densely, but we were able to increase the density slightly with the use of calcium chloride that promotes fusion of the vesicles. Finally, we used zwitterionic liposomes loaded with fluorescent probe calcein to show that they remain intact after the capture on the L1 chip. The fluorescence was lost only after we used equinatoxin, a well-studied pore-forming toxin, to perform on-chip permeabilization of vesicles. The characteristics of permeabilization process for chip-immobilized liposomes are similar to those of liposomes free in solution. All results collectively suggest that liposomes do not fuse to form a single bilayer on the surface of the chip.

Structural Analysis of the Laetiporus sulphureus Hemolytic Pore-forming Lectin in Complex with Sugars

LSL is a lectin produced by the parasitic mushroom Laetiporus sulphureus, which exhibits hemolytic and hemagglutinating activities. Here, we report the crystal structure of LSL refined to 2.6-A resolution determined by the single isomorphous replacement method with the anomalous scatter (SIRAS) signal of a platinum derivative. The structure reveals that LSL is hexameric, which was also shown by analytical ultracentrifugation. The monomeric protein (35 kDa) consists of two distinct modules: an N-terminal lectin module and a pore-forming module. The lectin module has a beta-trefoil scaffold that bears structural similarities to those present in toxins known to interact with galactose-related carbohydrates such as the hemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum, abrin, and ricin. On the other hand, the C-terminal pore-forming module (composed of domains 2 and 3) exhibits three-dimensional structural resemblances with domains 3 and 4 of the beta-pore-forming toxin aerolysin from the Gram-negative bacterium Aeromonas hydrophila, and domains 2 and 3 from the epsilon-toxin from Clostridium perfringens. This finding reveals the existence of common structural elements within the aerolysin-like family of toxins that could be directly involved in membrane-pore formation. The crystal structures of the complexes of LSL with lactose and N-acetyllactosamine reveal two dissacharide-binding sites per subunit and permits the identification of critical residues involved in sugar binding.

Pore Formation by Equinatoxin, a Eukaryotic Pore-forming Toxin, Requires a Flexible N-terminal Region and a Stable β-Sandwich

Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp117. These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.

Lipid Phase Coexistence Favors Membrane Insertion of Equinatoxin-II, a Pore-forming Toxin from Actinia equina

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 liquid-disordered 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.

A Novel Mechanism of Pore Formation: MEMBRANE PENETRATION BY THE N-TERMINAL AMPHIPATHIC REGION OF EQUINATOXIN

Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10-28 are organized as an alpha-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation.