The behavior of sea anemone actinoporins at the water-membrane interface

Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water-membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound.     

A Pore-Forming Toxin Requires a Specific Residue for Its Activity in Membranes with Particular Physicochemical Properties

The physicochemical landscape of the bilayer modulates membrane protein function. Actinoporins are a family of potent hemolytic proteins from sea anemones acting at the membrane level. This family of cytolysins preferentially binds to target membranes containing sphingomyelin, where they form lytic pores giving rise to cell death. Although the cytolytic activity of the actinoporin fragaceatoxin C (FraC) is sensitive to vesicles made of various lipid compositions, it is far from clear how this toxin adjusts its mechanism of action to a broad range of physiochemical landscapes. Herein, we show that the conserved residue Phe-16 of FraC is critical for pore formation in cholesterol-rich membranes such as those of red blood cells. The interaction of a panel of muteins of Phe-16 with model membranes composed of raft-like lipid domains is inactivated in cholesterol-rich membranes but not in cholesterol-depleted membranes. These results indicate that actinoporins recognize different membrane environments, resulting in a wider repertoire of susceptible target membranes (and preys) for sea anemones. In addition, this study has unveiled promising candidates for the development of protein-based biosensors highly sensitive to the concentration of cholesterol within the membrane.     

Characterization of the Lipid-Binding Site of Equinatoxin II by NMR and Molecular Dynamics Simulation

Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina. Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-¹⁵N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation.     

Solution structure of the eukaryotic pore-forming cytolysin equinatoxin II: implications for pore formation

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, that 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. The structure of the actinoporin equinatoxin II in aqueous solution, determined from NMR data, consists of two short helices packed against opposite faces of a beta-sandwich structure formed by two five-stranded beta-sheets. The protein core has extensive hydrophobic interfaces formed by residues projecting from the internal faces of the two beta-sheets. 15N relaxation data show uniform backbone dynamics, implying that equinatoxin II in solution is relatively rigid, except at the N terminus; its inferred rotational correlation time is consistent with values for monomeric proteins of similar mass. Backbone amide exchange rate data also support the view of a stable structure, even though equinatoxin II lacks disulfide bonds. As monitored by NMR, it unfolds at around 70 degrees C at pH 5.5. At 25 degrees C the structure is stable over the pH range 2.5-7.3 but below pH 2.5 it undergoes a slow transition to an incompletely unfolded structure resembling a molten globule. Equinatoxin II has two significant patches of positive electrostatic potential formed by surface-exposed Lys and Arg residues, which may assist its interaction with charged regions of the lipid head groups. Tyr and Trp residues on the surface may also contribute by interacting with the carbonyl groups of the acyl chains of target membranes. Data from mutational studies and truncated analogues identify two regions of the protein involved in membrane interactions, the N-terminal helix and the Trp-rich region. Once the protein is anchored, the N-terminal helix may penetrate the membrane, with up to four helices lining the pore, although other mechanisms of pore formation cannot be ruled out.     

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.     

Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins,pore-forming proteins from a sea anemone

Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that are able to bind and oligomerize in membranes, leading to cell swelling, impairment of ionic gradients and, eventually, to cell death. In this review we summarize the knowledge generated from the combination of biochemical and biophysical approaches to the study of sticholysins I and II (Sts, StI/II), two actinoporins largely characterized by the Center of Protein Studies at the University of Havana during the last 20 years. These approaches include strategies for understanding the toxin structure–function relationship, the protein–membrane association process leading to pore formation and the interaction of toxin with cells. The rational combination of experimental and theoretical tools have allowed unraveling, at least partially, of the complex mechanisms involved in toxin–membrane interaction and of the molecular pathways triggered upon this interaction. The study of actinoporins is important not only to gain an understanding of their biological roles in anemone venom but also to investigate basic molecular mechanisms of protein insertion into membranes, protein–lipid interactions and the modulation of protein conformation by lipid binding. A deeper knowledge of the basic molecular mechanisms involved in Sts–cell interaction, as described in this review, will support the current investigations conducted by our group which focus on the design of immunotoxins against tumor cells and antigen-releasing systems to cell cytosol as Sts-based vaccine platforms.     

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.     

Imaging the Lipid-Phase-Dependent Pore Formation of Equinatoxin II in Droplet Interface Bilayers

Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.     

Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier

To fulfil their function, nuclear pore complexes (NPCs) must discriminate between inert proteins and nuclear transport receptors (NTRs), admitting only the latter. This specific permeation is thought to depend on interactions between hydrophobic patches on NTRs and phenylalanine‐glycine (FG) or related repeats that line the NPC. Here, we tested this premise directly by conjugating different hydrophobic amino‐acid analogues to the surface of an inert protein and examining its ability to cross NPCs unassisted by NTRs. Conjugation of as few as four hydrophobic moieties was sufficient to enable passage of the protein through NPCs. Transport of the modified protein proceeded with rates comparable to those measured for the innate protein when bound to an NTR and was relatively insensitive both to the nature and density of the amino acids used to confer hydrophobicity. The latter observation suggests a non‐specific, small, and pliant interaction network between cargo and FG repeats.     

Imaging the Lipid-Phase-Dependent Pore Formation of Equinatoxin II in Droplet Interface Bilayers

Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.     

Toxicity of an α-Pore-forming Toxin Depends on the Assembly Mechanism on the Target Membrane as Revealed by Single Molecule Imaging

α-Pore-forming toxins (α-PFTs) are ubiquitous defense tools that kill cells by opening pores in the target cell membrane. Despite their relevance in host/pathogen interactions, very little is known about the pore stoichiometry and assembly pathway leading to membrane permeabilization. Equinatoxin II (EqtII) is a model α-PFT from sea anemone that oligomerizes and forms pores in sphingomyelin-containing membranes. Here, we determined the spatiotemporal organization of EqtII in living cells by single molecule imaging. Surprisingly, we found that on the cell surface EqtII did not organize into a unique oligomeric form. Instead, it existed as a mixture of oligomeric species mostly including monomers, dimers, tetramers, and hexamers. Mathematical modeling based on our data supported a new model in which toxin clustering happened in seconds and proceeded via condensation of EqtII dimer units formed upon monomer association. Furthermore, altering the pathway of EqtII assembly strongly affected its toxic activity, which highlights the relevance of the assembly mechanism on toxicity.     

The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition

The sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (St I and St II). Despite their high identity (93%), these toxins exhibit differences in hemolytic activity that can be related to those found in their N-terminal. To clarify the contribution of the N-terminal amino acid residues to the activity of the toxins, we synthesized peptides spanning residues 1-31 of St I (StI1-31) or 1-30 of St II (StII1-30) and demonstrated that StII1-30 promotes erythrocyte lysis to a higher extent than StI1-31. For a better understanding of the molecular mechanism underlying the peptide activity, here we studied their binding to lipid monolayers and pemeabilizing activity in liposomes. For this, we examined the effect on peptide membranotropic activity of including phospatidic acid and cholesterol in a lipid mixture of phosphatidylcholine and sphingomyelin. The results suggest the importance of continuity of the 1-10 hydrophobic sequence in StII1-30 for displaying higher binding and activity, in spite of both peptides' abilities to form pores in giant unilamellar vesicles. Thus, the different peptide membranotropic action is explained in terms of the differences in hydrophobic and electrostatic peptide properties as well as the enhancing role of membrane inhomogeneities.     

Two-step membrane binding by Equinatoxin II,a pore-forming toxin from the sea anemone,involves an exposed aromatic cluster and a flexible helix

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 membrane-embedded 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.     

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.     

Membrane Damage by an α-Helical Pore-forming Protein,Equinatoxin II,Proceeds through a Succession of Ordered Steps

Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from β-pore-forming toxins that often form β-barrel pores via a stable prepore complex.     

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.     

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.     

More Than a Pore: The Interplay of Pore-Forming Proteins and Lipid Membranes

Pore-forming proteins (PFPs) punch holes in their target cell membrane to alter their permeability. Permeabilization of lipid membranes by PFPs has received special attention to study the basic molecular mechanisms of protein insertion into membranes and the development of biotechnological tools. PFPs act through a general multi-step mechanism that involves (i) membrane partitioning, (ii) insertion into the hydrophobic core of the bilayer, (iii) oligomerization, and (iv) pore formation. Interestingly, PFPs and membranes show a dynamic interplay. As PFPs are usually produced as soluble proteins, they require a large conformational change for membrane insertion. Moreover, membrane structure is modified upon PFPs insertion. In this context, the toroidal pore model has been proposed to describe a pore architecture in which not only protein molecules but also lipids are directly involved in the structure. Here, we discuss how PFPs and lipids cooperate and remodel each other to achieve pore formation, and explore new evidences of protein-lipid pore structures.     

Ubiquitin‐like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway

Ubiquitin and ubiquitin‐like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre‐mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5‐deficient cells display spliceosome‐independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5–FANCI binding, we generated separation‐of‐function mutants to demonstrate that key aspects of FA pathway function, including FANCI–FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5–FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway.     

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.