Interaction of the eukaryotic pore-forming cytolysin equinatoxin II with model membranes: 19F NMR studies

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by site-directed mutagenesis. The 19F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingomyelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. The lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the beta-sandwich, which forms the bulk of the protein.     

Solid-state NMR study of membrane interactions of the pore-forming cytolysin, equinatoxin II

Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina that lyses red blood cell and model membranes. Lysis is dependent on the presence of sphingomyelin (SM) and is greatest for vesicles composed of equimolar SM and phosphatidylcholine (PC). Since SM and cholesterol (Chol) interact strongly, forming domains or “rafts” in PC membranes, ³¹P and ²H solid-state NMR were used to investigate changes in the lipid order and bilayer morphology of multilamellar vesicles comprised of different ratios of dimyristoylphosphatidylcholine (DMPC), SM and Chol following addition of EqtII. The toxin affects the phase transition temperature of the lipid acyl chains, causes formation of small vesicle type structures with increasing temperature, and changes the T₂ relaxation time of the phospholipid headgroup, with a tendency to order the liquid disordered phases and disorder the more ordered lipid phases. The solid-state NMR results indicate that Chol stabilizes the DMPC bilayer in the presence of EqtII but leads to greater disruption when SM is in the bilayer. This supports the proposal that EqtII is more lytic when both SM and Chol are present as a consequence of the formation of domain boundaries between liquid ordered and disordered phases in lipid bilayers leading to membrane disruption.     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Structure and activity of the N-terminal region of the eukaryotic cytolysin equinatoxin II

The actinoporins are a family of proteins from sea anemones that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being resistant to these toxins. Pore formation by the actinoporin equinatoxin II (EqTII) proceeds by membrane binding via a surface rich in aromatic residues, followed by translocation of the N-terminal region to the membrane and, finally, across the bilayer to form a functional pore. A key feature of this mechanism is the ability of the N-terminal region to form a stable, bilayer-spanning helix in the membrane, which in turn requires dissociation of the N-terminus from the bulk of the protein and significant extension of the N-terminal helix of native EqTII. In this study the structures of three peptides corresponding to residues 11-29, 11-32, and 1-32, respectively, of EqTII have been investigated by high-resolution nuclear magnetic resonance and Fourier transform infrared spectroscopy. The 32-residue peptide lacks ordered secondary structure in water, but residues 6-28 form a helix in dodecylphosphocholine micelles. Although this helix is long enough to span a bilayer membrane, this peptide and the shorter analogues display limited permeabilizing activity in large unilamellar vesicles and very weak hemolytic activity in human red blood cells. Thus, while the N-terminal region has the structural features required for this unusual mechanism of pore formation, the lack of activity of the isolated N-terminus shows that the bulk of the protein is essential for efficient pore formation by facilitating initial membrane binding, interacting with sphingomyelin, or stabilizing the oligomeric pore.     

Pre-pore oligomer formation by Vibrio cholerae cytolysin: Insights from a truncated variant lacking the pore-forming pre-stem loop

Vibrio cholerae cytolysin (VCC), a β-barrel pore-forming toxin (β-PFT), induces killing of the target eukaryotic cells by forming heptameric transmembrane β-barrel pores. Consistent with the β-PFT mode of action, binding of the VCC toxin monomers with the target cell membrane triggers formation of pre-pore oligomeric intermediates, followed by membrane insertion of the β-strands contributed by the pre-stem motif within the central cytolysin domain of each protomer. It has been shown previously that blocking of membrane insertion of the VCC pre-stem motif arrests conversion of the pre-pore state to the functional transmembrane pore. Consistent with the generalized β-PFT mechanism, it therefore appears that the VCC pre-stem motif plays a critical role toward forming the structural scaffold of the transmembrane β-barrel pore. It is, however, still not known whether the pre-stem motif plays any role in the membrane interaction process, and subsequent pre-pore structure formation by VCC. In this direction, we have constructed a recombinant variant of VCC deleting the pre-stem region, and have characterized the effect(s) of physical absence of the pre-stem motif on the distinct steps of the membrane pore-formation process. Our results show that the deletion of the pre-stem segment does not affect membrane binding and pre-pore oligomer formation by the toxin, but it critically abrogates the functional pore-forming activity of VCC. Present study extends our insights regarding the structure–function mechanism associated with the membrane pore formation by VCC, in the context of the β-PFT mode of action.     

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: A synchrotron radiation circular dichroism spectroscopic study

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the ‘bound’ state and the ‘free’ state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.     

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: a synchrotron radiation circular dichroism spectroscopic study

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the 'bound' state and the 'free' state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.     

Solution structure of APETx1 from the sea anemone Anthopleura elegantissima: a new fold for an HERG toxin

APETx1 is a 42-amino acid toxin purified from the venom of the sea anemone Anthopleura elegantissima. This cysteine-rich peptide possesses three disulfide bridges (C4-C37, C6-C30, and C20-C38). Its pharmacological target is the Ether-a-gogo potassium channel. We herein determine the solution structure of APETx1 by use of conventional two-dimensional 1H-NMR techniques followed by torsion angle dynamics and refinement protocols. The calculated structure of APETx1 belongs to the disulfide-rich all-beta structural family, in which a three-stranded anti-parallel beta-sheet is the only secondary structure. APETx1 is the first Ether-a-gogo effector discovered to fold in this way. We therefore compare the structure of APETx1 to those of the two other known effectors of the Ether-a-gogo potassium channel, CnErg1 and BeKm-1, and analyze the topological disposition of key functional residues proposed by analysis of the electrostatic anisotropy. The interacting surface is made of a patch of aromatic residues (Y5, Y32, and F33) together with two basic residues (K8 and K18) at the periphery of the surface. We pinpoint the absence of the central lysine present in the functional surface of the two other Ether-a-gogo effectors.     

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.     

Induction of nitric oxide synthase expression by Vibrio vulnificus cytolysin.

The pore-forming cytolysin of Vibrio vulnificus (VVC) causes severe hypotension and vasodilatation in vivo. Under the condition of bacterial sepsis, large amounts of nitric oxide (NO) produced by inducible NO synthase (iNOS) can contribute to host-induced tissue damage causing hypotension and septic shock. In this study, we investigated the effect of purified VVC on NO production in mouse peritoneal macrophages. VVC induced NO production in the presence of interferon-gamma. Increased NO production was not affected by polymyxin B, and heat inactivation of cytolysin abolished the NO-inducing capability. NO production was induced at the same concentration range of cytolysin for pore formation, as evidenced by the release of preloaded 2-deoxy-d-[(3)H]glucose. At the higher concentrations of cytolysin causing the depletion of cellular ATP, no NO production was observed. Increased expression of iNOS and activation of NFkappaB by VVC were confirmed by Western blotting and gel shift assay, respectively. These results suggest the role of cytolysin as an inducer of iNOS and NO production in macrophage and as a possible virulence determinant in V. vulnificus infection.     

Cytotoxicity of equinatoxin II from the sea anemoneActinia equina involves ion channel formation and an increase in intracellular calcium activity

Summary Equinatoxin Il is a 20-kDa basic protein isolated from the sea anemoneActinia equina. The aim of our work was to investigate the primary molecular basis for the cytotoxic effects of equinatoxin II in two model systems: single bovine lactotrophs and planar lipid bilayers. Previous work has shown that equinatoxin II produces rapid changes in cell morphology, which are dependent on external calcium. It has also been reported that addition of equinatoxin II increases membrane electrical conductance, which suggests that the cytotoxic action of equinatoxin II involves an increase in the permeability of membranes to Ca²⁺. Extensive changes in cytosolic Ca²⁺ activity are thought to invoke irreversible changes in cell physiology and morphology. In this paper, we show that morphological changes brought about by equinatoxin II in bovine lactotrophs are associated with a rapid rise in cytosolic Ca²⁺ activity, monitored with a fura-2 video imaging apparatus. Moreover, incorporation of equinatoxin II into planar lipid bilayers produces Ca²⁺ permeable ion channels. This suggests that the mode of equinatoxin II cytotoxicity involves the formation of cation (Ca²⁺) permeable channels in cell membranes.     

Three-dimensional structure of the neurotoxin ATX Ia from Anemonia sulcata in aqueous solution determined by nuclear magnetic resonance spectroscopy

With the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three-dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and chi 1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sampling of all conformations that are consistent with the local and medium-range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of beta-methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 +/- 0.3 A. This core is formed by a four-stranded beta-sheet connected by two well-defined loops, and there is an additional flexible loop consisting of the eleven residues 8-18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues and shielded on one side by hydrophilic residues.     

Vertical collapse of a cytolysin prepore moves its transmembrane beta-hairpins to the membrane

Perfringolysin O (PFO) is a prototype of the large family of pore-forming cholesterol-dependent cytolysins (CDCs). A central enigma of the cytolytic mechanism of the CDCs is that their membrane-spanning beta-hairpins (the transmembrane amphipathic beta-hairpins (TMHs)) appear to be approximately 40 A too far above the membrane surface to cross the bilayer and form the pore. We now present evidence, using atomic force microscopy (AFM), of a significant difference in the height by which the prepore and pore protrude from the membrane surface: 113+/-5 A for the prepore but only 73+/-5 A for the pore. Time-lapse AFM micrographs show this change in height in real time. Moreover, the monomers in both complexes exhibit nearly identical surface features and these results in combination with those of spectrofluorimetric analyses indicate that the monomers remain in a perpendicular orientation to the bilayer plane during this transition. Therefore, the PFO undergoes a vertical collapse that brings its TMHs to the membrane surface so that they can extend across the bilayer to form the beta-barrel pore.     

Crystal structure of the Vibrio cholerae cytolysin (VCC) pro-toxin and its assembly into a heptameric transmembrane pore

Pathogenic Vibrio cholerae secrete V. cholerae cytolysin (VCC), an 80 kDa pro-toxin that assembles into an oligomeric pore on target cell membranes following proteolytic cleavage and interaction with cell surface receptors. To gain insight into the activation and targeting activities of VCC, we solved the crystal structure of the pro-toxin at 2.3A by X-ray diffraction. The core cytolytic domain of VCC shares a fold similar to the staphylococcal pore-forming toxins, but in VCC an amino-terminal pro-domain and two carboxy-terminal lectin domains decorate the cytolytic domain. The pro-domain masks a protomer surface that likely participates in inter-protomer interactions in the cytolytic oligomer, thereby explaining why proteolytic cleavage and movement of the pro-domain is necessary for toxin activation. A single beta-octyl glucoside molecule outlines a possible receptor binding site on one lectin domain, and removal of this domain leads to a tenfold decrease in lytic activity toward rabbit erythrocytes. VCC activated by proteolytic cleavage assembles into an oligomeric species upon addition of soybean asolectin/cholesterol liposomes and this oligomer was purified in detergent micelles. Analytical ultracentrifugation and crystallographic analysis indicate that the resulting VCC oligomer is a heptamer. Taken together, these studies define the architecture of a pore forming toxin and associated lectin domains, confirm the stoichiometry of the assembled oligomer as heptameric, and suggest a common mechanism of assembly for staphylococcal and Vibrio cytolytic toxins.     

Crystal structure of the soluble form of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina

BACKGROUND: Membrane pore-forming toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a membrane, undergoes a series of transformations, leading to an active, membrane-bound form. In contrast to bacterial toxins, no structure of a pore-forming toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinatoxin II (EqtII) may unravel a novel mechanism. RESULTS: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand beta sandwich fold with a hydrophobic core and a pair of alpha helices, each of which is associated with the face of a beta sheet. CONCLUSIONS: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the beta sandwich core. This segment includes a three-turn alpha helix that lies on the surface of a beta sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the membrane pore, whereas the beta sandwich structure remains unaltered and attaches to a membrane as do other structurally related extrinsic membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in membrane pore formation.     

Solution structure of APETx2, a specific peptide inhibitor of ASIC3 proton-gated channels

Acid-sensing ion channels (ASIC) are proton-gated sodium channels that have been implicated in pain transduction associated with acidosis in inflamed or ischemic tissues. APETx2, a peptide toxin effector of ASIC3, has been purified from an extract of the sea anemone Anthopleura elegantissima. APETx2 is a 42-amino-acid peptide cross-linked by three disulfide bridges. Its three-dimensional structure, as determined by conventional two-dimensional 1H-NMR, consists of a compact disulfide-bonded core composed of a four-stranded beta-sheet. It belongs to the disulfide-rich all-beta structural family encompassing peptide toxins commonly found in animal venoms. The structural characteristics of APETx2 are compared with that of PcTx1, another effector of ASIC channels but specific to the ASIC1a subtype and to APETx1, a toxin structurally related to APETx2, which targets the HERG potassium channel. Structural comparisons, coupled with the analysis of the electrostatic characteristics of these various ion channel effectors, led us to suggest a putative channel interaction surface for APETx2, encompassing its N terminus together with the type I-beta turn connecting beta-strands III and IV. This basic surface (R31 and R17) is also rich in aromatic residues (Y16, F15, Y32, and F33). An additional region made of the type II'-beta turn connecting beta-strands I and II could also play a role in the specificity observed for these different ion effectors.     

Bacterial protein toxins and lipids: pore formation or toxin entry into cells

Lipids are hydrophobic molecules which play critical functions in cells, in particular, they are essential constituents of membranes, whereas bacterial toxins are mainly hydrophilic proteins. All bacterial toxins interact first with their target cells by recognizing a surface receptor, which is either a lipid or a lipid derivative, or another compound but in a lipid environment. Most bacterial toxins are PFTs (pore-forming toxins) which oligomerize and insert into the lipid bilayer. A common mechanism of action involves the formation of a beta-barrel structure, resulting from the assembly of individual beta-hairpin(s) from individual monomers. An essential step for intracellular active toxins is to translocate their enzymatic part into the cytosol. Some toxins use a translocation mechanism based on pore formation similar to that of PFTs, others undergo a yet unclear 'chaperone' process.     

Asymmetric formation and possible function of the primary pore canal in plutei of Temnopleurus hardwicki

The development and possible function of the primary pore canal (PPC) in plutei of the sea urchin Temnopleurus hardwicki was examined by immunochemistry, electron microscopy and microsurgery. Left and right PPC that extended from coelomic sacs in plutei contained a bundle of cilia with a 9 + 2 structure that was initially detected as a group of anti-acetylated tubulin antibody-binding granules in the epithelium of coelomic sacs in 28 h postfertilization (PF) prism larvae. The granules extended to be a bundle of fibers toward the larval dorsal surface, concurrent with formation of the PPC on both sides, over the next 4 h. The cilia in both PPC beat actively. However, the PPC on the right side disappeared by approximately 55 h PF, establishing left-right asymmetry by 60 h PF (the four-arm pluteus stage). The numbers of cilia in the left and right PPC in 56 h PF plutei were five and eight, respectively. Microsurgical removal of the coelomic sac from both sides or the left side only from 26 h PF prism larvae decreased body width to 64 and 91% of normal width by 50 h PF pluteus stage, respectively, whereas that of the right PPC did not. These observations suggest that PPC contribute to the maintenance of normal body width, and that there is asymmetrical activity between the left and right PPC.