1.C.38 The Pore-forming Equinatoxin (Equinatoxin) Family

Sea anemones such as Actinia equina, Heteractis magnifica, and Stichodactyla helianthus produce a variety of sequence related toxins (called actinoporins) including Equinatoxins 1A,-1D, II, III, IV, V, etc. They have been given alternative designations such as Tenebrosin C (for Equinatoxin II), cytolysin, and hemolytic toxin. These cardiac stimulatory hemolysins penetrate membranes forming ion permeable, cation-selective pores, also permeable to small neutral solutes. They cause a variety of phenotypes in mammals including platelet aggregation, cytotoxicity of a variety of animal cells, lysis of some cells, and vasospasm of coronary vessels. They are of 175-179 aas in length and form tetrameric pore-forming structures in membranes. The 3-dimensional structure of the soluble form of equinatoxin II has been solved (Anastasiadis et al., 2001). The radius of the Sticholysin pore has been shown to be about 1.2 nm (diameter, ~2 nm). Pore size is independent of toxin concentration and is the same in biological and artificial membranes (Tejuca et al., 2001). Pore formation requires a flexible N-terminal region and a stable β-sandwich (Kristan et al., 2004). Actinoporins comprise a multigene family consisting of 47 representatives expressed in the sea anemone tentacles as prepropeptide-coding transcripts. Phylogenetic analysis revealed that actinoporin clustering is consistent with the division of sea anemones into superfamilies and families (Leychenko et al. 2018).  The structural mechanisms of pore formation and host-pathogen interaction of PFTs at an atomic level have been reviewed (Li et al. 2021).

Equinatoxin II inserts into the membrane via a two-step membrane-binding process involving an exposed cluster of aromatic residues (step 1) and a flexible N-terminal amphipathic α-helix (step 2) (Hong et al., 2002). The first step is similar to that of the evolutionarily distant cholesterol-dependent cytolysins. Interaction is dependent on sphingomyelin, and lipid phase coexistence favors membrane insertion (Barlic et al., 2004; Biserka et al., 2008; Schoen et al., 2008).

Equinatoxin II (EqtII) from Actinia equina and Sticholysin II (StnII) from Stichodactyla helianthus are the actinoporins that have been studied in greatest detail. Both proteins display a beta-sandwich fold composed of 10 β-strands flanked on each side by two short alpha-helices. Two-dimensional crystallization on lipid monolayers has allowed the determination of low-resolution models of tetrameric structures distinct from the pore. Wild-type EqtII and StnII, as well as a nice collection of natural and artificially made variants of both proteins, have been produced in Escherichia coli and purified. Four regions of the actinoporin structure seem to play an important role. The phosphatidyl choline or sphingomyelin-binding site and a cluster of exposed aromatic residues, together with a basic region, may be involved in the initial interaction with the membrane, whereas the amphipathic N-terminal region is essential for oligomerization and pore formation (Alegre-Cebollaba et al., 2007). Pore formation proceeds in at least four steps: Monomer binding to the membrane interface, assembly of four monomers, and at least two distinct conformational changes driving to the final formation of the functional pore. Sticholysin I is almost identical to sticholysin II. Conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation (Alegre-Cebollada et al., 2008). An AF domain superfamily (abbreviated from actinoporin-like proteins and fungal fruit-body lectins) has been defined. It contains members from at least three animal and two plant phyla. On the basis of functional properties of some members, Crnigoj Kristan et al., 2009 hypothesised that AF domains mediate peripheral membrane interactions.

Fragaceatoxin C (FraC) is an α-barrel pore-forming toxin (PFT). The crystal structures of FraC at four different stages of the lytic mechanism have been determined at 3.1Å resolution, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore (Tanaka et al. 2015). 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 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 this haemolytic toxin.

The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate is known as a prepore. Morante et al. 2016 employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) to identify a prepore species of the actinoporin fragaceatoxin C (FraC) (TC# 1.C.38.1.12) bound to lipid vesicles. The size of the prepore coincides with that of the functional pore except for the transmembrane region, which is absent in the prepore. In the prepore species, the N-terminus is not inserted in the bilayer but is exposed to the aqueous solution. 

Sea anemones (Cnidaria, Anthozoa, and Actiniaria) use toxic peptides to incapacitate and immobilize prey and to deter potential predators. Their toxin arsenal is complex, targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Among these, actinoporins are one of the better characterized toxins; these venom proteins form a pore in cellular membranes containing sphingomyelin. Macrander and Daly 2016 used a combined bioinformatic and phylogenetic approach to investigate how actinoporins have evolved across three superfamilies of sea anemones (Actinioidea, Metridioidea, and Actinostoloidea). Their analysis identified 90 candidate actinoporins across 20 species. They also found clusters of six actinoporin-like genes in five species of sea anemone (Nematostella vectensis, Stomphia coccinea, Epiactis japonica, Heteractis crispa, and Diadumene leucolena); these actinoporin-like sequences resembled actinoporins but have a higher sequence similarity with toxins from fungi, cone snails, and Hydra. Comparative analysis of the candidate actinoporins highlighted variable and conserved regions within actinoporins that may pertain to functional variation.Multiple residues are involved in initiating sphingomyelin recognition and membrane binding. Residues thought to be involved with oligomerization were variable, while those forming the phosphocholine (POC) binding site and the N-terminal region involved with cell membrane penetration were highly conserved (Macrander and Daly 2016).

Equinatoxin II (EqtII), fragaceatoxin C (FraC), and sticholysins I and II (StnI and StnII, respectively), produced by three different sea anemone species, are the only actinoporins whose molecular structures had been studied in depth as of Jan, 2017. These four proteins show high sequence identities and practically coincident three-dimensional structures, but, their pore-forming activities are quite different depending on the model lipid system employed (García-Linares et al. 2016). These varied responses to lipid composition may be a consequence of their distinct specificities and/or membrane binding affinities. Trp residues play a major role in membrane recognition and binding, but these residues have only a minor influence on the diffusion and oligomerization steps needed to assemble a functional pore (García-Linares et al. 2016).

The generalized transport reaction catalyzed by members of the equinatoxin family is:

Small molecule (in) small molecule (out)


 

References:

Alegre-Cebollada, J., M. Cunietti, E. Herrero-Galán, J.G. Gavilanes, and A. Martínez-del-Pozo. (2008). Calorimetric scrutiny of lipid binding by sticholysin II toxin mutants. J. Mol. Biol. 382: 920-930.

Alegre-Cebollada, J., M. Oñaderra, J.G. Gavilanes, and A.M. del Pozo. (2007). Sea anemone actinoporins: the transition from a folded soluble state to a functionally active membrane-bound oligomeric pore. Curr. Protein. Pept. Sci. 8: 558-572.

Alvarado-Mesén, J., F. Solano-Campos, L. Canet, L. Pedrera, Y.P. Hervis, C. Soto, H. Borbón, M.E. Lanio, B. Lomonte, A. Valle, and C. Alvarez. (2019). Cloning, purification and characterization of nigrelysin, a novel actinoporin from the sea anemone Anthopleura nigrescens. Biochimie 156: 206-223.

Alvarez C., Mancheno JM., Martinez D., Tejuca M., Pazos F. and Lanio ME. (2009). Sticholysins, two pore-forming toxins produced by the Caribbean Sea anemone Stichodactyla helianthus: their interaction with membranes. Toxicon. 54(8):1135-47.

Anastasiadis, A., G. Anderluh, P. Maeek, and D. Turk. (2001). Crystal structure of the soluble form of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina. Structure 9: 341-346.

Bakrac, B., A. Kladnik, P. Macek, G. McHaffie, A. Werner, J.H. Lakey, and G. Anderluh. (2010). A toxin-based probe reveals cytoplasmic exposure of Golgi sphingomyelin. J. Biol. Chem. 285: 22186-22195.

Bakrac, B., I. Gutiérrez-Aguirre, Z. Podlesek, A.F. Sonnen, R.J. Gilbert, P. Macek, J.H. Lakey, and G. Anderluh. (2008). Molecular determinants of sphingomyelin specificity of a eukaryotic pore-forming toxin. J. Biol. Chem. 283: 18665-18677.

Barlic, A., I. Gutiérrez-Aguirre, J.M.M. Caaveiro, A. Cruz, M.-B. Ruiz-Argüello, J. Pérez-Gil, and J.M. González-Mañas. (2004). Lipid phase coexistence favors membrane insertion of equinatoxin-II, a pore-forming toxin from Actinia equina. J. Biol. Chem. 279: 34209-34216.

Belmonte, G., G. Menestrina, C. Pederzolli, I. Kriaj, F. Gubensek, T. Turk, and P. Macek. (1994). Primary and secondary structure of a pore-forming toxin from the sea anemone, Actinia equina L, and its association with lipid vesicles. Biochim. Biophys. Acta 1192: 197-204.

Borges, M.H., F. Andrich, P.H. Lemos, T.G. Soares, T.N. Menezes, F.V. Campos, L.X. Neves, W. Castro-Borges, and S.G. Figueiredo. (2018). Combined proteomic and functional analysis reveals rich sources of protein diversity in skin mucus and venom from the Scorpaena plumieri fish. J Proteomics 187: 200-211.

Frazão, B., V. Vasconcelos, and A. Antunes. (2012). Sea anemone (Cnidaria, Anthozoa, Actiniaria) toxins: an overview. Mar Drugs 10: 1812-1851.

García-Linares, S., E. Rivera-de-Torre, K. Morante, K. Tsumoto, J.M. Caaveiro, J.G. Gavilanes, J.P. Slotte, and &.#.1.9.3.;. Martínez-Del-Pozo. (2016). Differential Effect of Membrane Composition on the Pore-Forming Ability of Four Different Sea Anemone Actinoporins. Biochemistry 55: 6630-6641.

García-Linares, S., T. Maula, E. Rivera-de-Torre, J.G. Gavilanes, J.P. Slotte, and &.#.1.9.3.;. Martínez-Del-Pozo. (2016). Role of the Tryptophan Residues in the Specific Interaction of the Sea Anemone Stichodactyla helianthus's Actinoporin Sticholysin II with Biological Membranes. Biochemistry 55: 6406-6420.

Gupta, L.K., J. Molla, and A.A. Prabhu. (2023). Story of Pore-Forming Proteins from Deadly Disease-Causing Agents to Modern Applications with Evolutionary Significance. Mol Biotechnol. [Epub: Ahead of Print]

Hoang, Q.T., S.H. Cho, S.F. McDaniel, S.H. Ok, R.S. Quatrano, and J.S. Shin. (2009). An actinoporin plays a key role in water stress in the moss Physcomitrella patens. New Phytol 184: 502-510.

Hong, Q. I. Gutiérrez-Aguirre, A. Barlic, P. Malovrh, K. Kristan, Z. Podlesek, P. Macek, D. Turk, J.M. Gonzáles-Mañas, J.H. Lakey, and G. Anderluh. (2002). Two-step membrane binding by equinatoxin II, a pore-forming toxin from the sea anemone, involves an exposed aromatic cluster and a flexible helix. J. Biol. Chem. 277: 41916-41924.

Huang, G., K. Willems, M. Soskine, C. Wloka, and G. Maglia. (2017). Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores. Nat Commun 8: 935.

Kawashima Y., H. Nagai, M. Ishida, Y. Nagashima, K. Shiomi. (2003). Primary structure of echotoxin 2, an actinoporin-like hemolytic toxin from the salivary gland of the marine gastropod Monoplex echo. Toxicon. 42: 491-497.

Kristan KC., Viero G., Dalla Serra M., Macek P. and Anderluh G. (2009). Molecular mechanism of pore formation by actinoporins. Toxicon. 54(8):1125-34.

Kristan, K., Z. Podlesek, V. Hojnik, I. Gutiérrez-Aguirre, G. Guncar, D. Turk, J.M. González-Mañas, J.H. Lakey, P. Macek, and G. Anderluh. (2004). Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable β-sandwich. J. Biol. Chem. 279: 46509-46517.

Lanio, M.E., V. Morera, C. Alvarez, M. Tejuca, T. Gómez, F. Pazos, V. Besada, D. Martínez, V. Huerta, G. Padrón, and M. de los Angeles Chávez. (2001). Purification and characterization of two hemolysins from Stichodactyla helianthus. Toxicon 39: 187-194.

Leychenko, E., M. Isaeva, E. Tkacheva, E. Zelepuga, A. Kvetkina, K. Guzev, M. Monastyrnaya, and E. Kozlovskaya. (2018). Multigene Family of Pore-Forming Toxins from Sea Anemone. Mar Drugs 16:.

Li, Y., Y. Li, H.M. Mengist, C. Shi, C. Zhang, B. Wang, T. Li, Y. Huang, Y. Xu, and T. Jin. (2021). Structural Basis of the Pore-Forming Toxin/Membrane Interaction. Toxins (Basel) 13:.

Macrander, J. and M. Daly. (2016). Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones. Toxins (Basel) 8:.

Mebs D., Langeluddeke T. (1992). European viper venoms: haemorrhagic and myotoxic activities. Toxicon. 30: 1303-1306.

Mechaly, A.E., A. Bellomio, D. Gil-Cartón, K. Morante, M. Valle, J.M. González-Mañas, and D.M. Guérin. (2011). Structural insights into the oligomerization and architecture of eukaryotic membrane pore-forming toxins. Structure 19: 181-191.

Mesa-Galloso, H., K.H. Delgado-Magnero, S. Cabezas, A. López-Castilla, J.E. Hernández-González, L. Pedrera, C. Alvarez, D.P. Tieleman, A.J. García-Saez, M.E. Lanio, U. Ros, and P.A. Valiente. (2016). Disrupting a key hydrophobic pair in the oligomerization interface of the actinoporins impairs their pore-forming activity. Protein. Sci. [Epub: Ahead of Print]

Mesa-Galloso, H., P.A. Valiente, M.E. Valdés-Tresanco, R.F. Epand, M.E. Lanio, R.M. Epand, C. Alvarez, D.P. Tieleman, and U. Ros. (2019). Membrane Remodeling by the Lytic Fragment of SticholysinII: Implications for the Toroidal Pore Model. Biophys. J. [Epub: Ahead of Print]

Morante, K., A. Bellomio, D. Gil-Carton, L. Redondo-Morata, J. Sot, S. Scheuring, M. Valle, J.M. Gonzalez-Manas, K. Tsumoto, and J.M. Caaveiro. (2016). Identification of a Membrane-Bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins. J. Biol. Chem. [Epub: Ahead of Print]

Morante, K., J.M. Caaveiro, A.R. Viguera, K. Tsumoto, and J.M. González-Mañas. (2015). Functional characterization of Val60, a key residue involved in the membrane-oligomerization of fragaceatoxin C, an actinoporin from Actinia fragacea. FEBS Lett. 589: 1840-1846.

Poklar, N., J. Fritz, P. Macek, G. Vesnaver, and T.V. Chalikian. (1999). Interaction of the pore-forming protein equinatoxin II with model lipid membranes: a calorimetric and spectroscopic study. Biochemistry 38: 14999-15008.

Rojko, N., M. Dalla Serra, P. Maček, and G. Anderluh. (2015). Pore formation by actinoporins, cytolysins from sea anemones. Biochim. Biophys. Acta. [Epub: Ahead of Print]

Ros, U., G.P.B. Carretero, J. Paulino, E. Crusca, Jr, F. Pazos, E.M. Cilli, M.E. Lanio, S. Schreier, and C. Alvarez. (2019). Self-association and folding in membrane determine the mode of action of peptides from the lytic segment of sticholysins. Biochimie 156: 109-117.

Schön, P., A.J. García-Sáez, P. Malovrh, K. Bacia, G. Anderluh, and P. Schwille. (2008). Equinatoxin II permeabilizing activity depends on the presence of sphingomyelin and lipid phase coexistence. Biophys. J. 95: 691-698.

Shiomi K., Y. Kawashima, M. Mizukami, Y. Nagashima. (2002). Properties of proteinaceous toxins in the salivary gland of the marine gastropod (Monoplex echo). Toxicon. 40: 563-71.

Simpson, R.J., G.E. Reid, R.L. Maritz, C. Morton, and R.S. Norton. (1990). Complete amino acid sequence of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone Actina tenebrosa. Eur. J. Biochem. 190: 319-328.

Šolinc, G., T. Švigelj, N. Omersa, T. Snoj, K. Pirc, N. Žnidaršič, A. Yamaji-Hasegawa, T. Kobayashi, G. Anderluh, and M. Podobnik. (2022). Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians. J. Biol. Chem. 298: 102455.

Soto, C., G. Bergado, R. Blanco, T. Griñán, H. Rodríguez, U. Ros, F. Pazos, M.E. Lanio, A.M. Hernández, and C. Álvarez. (2018). Sticholysin II-mediated cytotoxicity involves the activation of regulated intracellular responses that anticipates cell death. Biochimie 148: 18-35.

Tanaka, K., J.M. Caaveiro, K. Morante, J.M. González-Mañas, and K. Tsumoto. (2015). Structural basis for self-assembly of a cytolytic pore lined by protein and lipid. Nat Commun 6: 6337.

Tejuca, M., S.M. Dalla, C. Potrich, C. Alvarez, and G. Menestrina. (2001). Sizing and radius of the pore formed in erythrocytes and lipid vesicles by the toxin sticholysin I from the sea anemone Stichodactyla helianthus. J. Membr. Biol. 183: 125-135.

Wloka, C., N.L. Mutter, M. Soskine, and G. Maglia. (2016). Alpha-Helical Fragaceatoxin C Nanopore Engineered for Double-Stranded and Single-Stranded Nucleic Acid Analysis. Angew Chem Int Ed Engl 55: 12494-12498.

Examples:

TC#NameOrganismal TypeExample
1.C.38.1.1

Equinatoxin II (EqtII) binds sphingomyelin specifically and localizes to the Golgi apparatus (Bakrac et al., 2010).  Disrupting the key hydrophobic interaction between V60 and F163 (FraC numbering scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII) and sticholysin II (StII) impairs the pore formation activity (Mesa-Galloso et al. 2016). Reviewed by Gupta et al. 2023.

Animals

Equinatoxin of Actinia tenebrosa (P61915)

 
1.C.38.1.10

Cytolysin RTX-A of 175 aas. Forms cations-selective hydrophilic pores of around 1 nm and causes cardiac stimulation and hemolysis. Pore formation is a multi-step process that involves specific recognition of membrane sphingomyelin (but neither cholesterol nor phosphatidylcholine) and requires oligomerization of the toxin subunits (Frazão et al. 2012). 

Animals

Cytolysin RTX-A of Heteactis crispa (Radianthus macrodactylus) (Leathery sea anemone)

 
1.C.38.1.11

Cytolysin Src-1 of 216 aas (Frazão et al. 2012).

Animals

Cytolysin Src-1 of Sagartia rosea (sea anemone)

 
1.C.38.1.12

Fragaceatoxin C (FraC), an alpha-barrel pore-forming protein, a cytolytic actinoporin, of 152 aas (Morante et al. 2015; Rojko et al. 2015).  Pore formation goes through a dimer intermediate and then generates the active octamer. Disrupting the key hydrophobic interaction between V60 and F163 (FraC numbering scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII) and sticholysin II (StII) impairs the pore formation activity (Mesa-Galloso et al. 2016).

Animals

FraC of Callorhynchus milii

 
1.C.38.1.13

Conoporin 1 of 242 aas

Animals

Conotoxin 1 of Conus geographus (Geography cone) (Nubecula geographus)

 
1.C.38.1.14

Pore-forming toxin, Nigrelysin of 214 aas. The toxin lacks Cys and readily permeabilizes erythrocytes, as well as L1210 cells. CD spectroscopy revealed that its secondary structure is dominated by beta structure (58.5%) with 5.5% α-helix, and 35% random structure. Binding experiments to lipidic monolayers and to liposomes, as well as permeabilization studies in vesicles, revealed that the affinity of this toxin for sphingomyelin-containing membranes is quite similar to sticholysin II (StII) (Alvarado-Mesén et al. 2019).

Nigrelysin of Anthopleura nigrescens

 
1.C.38.1.15

Pore-forming cytolysin, Src-1-like, isoform X1 of 225 aas (Borges et al. 2018).

Cytolysin of Notothenia coriiceps

 
1.C.38.1.16

Bryoporin of 178 aas, possibly with an N-terminal TMS.  It has hemolytic activity in vitro and probably binds a phosphocholine derivative with the unique amido or hydroxyl groups found in sphingomyelin. It is involved in drought tolerance and is inhibited by sphingomyelin (Hoang et al. 2009). Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians (Šolinc et al. 2022).

Bryoporin of Physcomitrium pates (spreading leaved earth moss) (Physcomitrella patens)

 
1.C.38.1.17

Sticholysin II, EstII; She4, of 175 aas. Its 3-D structure has been solved (1GWY).

She4 of Stichodactyla helianthus

 
1.C.38.1.18

Hydra Actinoporin-like toxin 1 of 187 aas. The 3-D structure is known (PDB acc# 7EKZ).

Toxin of Hydra vulgaris (Hydra attenuata)

 
1.C.38.1.2

Sticholysin I (cytolysin ST1; STNI STII; StiII; FraC) (Alvarez et al., 2009). Pore formation goes through a dimer intermediate and then generates the active octamer. Disrupting the key hydrophobic interaction between V60 and F163 (FraC numbering scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII) and sticholysin II (StII) impairs the pore formation activity (Mesa-Galloso et al. 2016). Sticholysin II-mediated cytotoxicity may involve the activation of regulated intracellular responses that anticipates cell death (Soto et al. 2018). Sticholysins represent a prototype of proteins acting through the formation of protein-lipid toroidal pores. Peptides spanning the N-terminus of sticholysins mimic the permeabilizing activity of the full-length toxins (Mesa-Galloso et al. 2019). Phospholipids integrate into the ring of the toroidal pores, promoting their stabilization. Self-association and folding in the membrane determine the mode of action of peptides from the lytic segment of sticholysins (Ros et al. 2019). STNI and STNII are 94% identical. They form cations-selective hydrophilic pores of around 1 nm and causes cardiac stimulation and cytolysis. Lanio et al. 2001 showed that pore formation is a multi-step process that involves specific recognition of membrane sphingomyelin (but neither cholesterol nor phosphatidylcholine) using an aromatic rich region and an adjacent phosphocholine (POC) binding site, firm binding to the membrane (mainly driven by hydrophobic interactions) accompanied by the transfer of the N-terminal region to the lipid-water interface and finally pore formation after oligomerization of monomers. Cytolytic effects include red blood cell hemolysis, platelet aggregation and lysis, cytotoxic and cytostatic effects on fibroblasts. Lethality in mammals has been ascribed to severe vasospasm of coronary vessels, cardiac arrhythmia, and inotropic effects (Lanio et al. 2001).

Animals

Sticholysin I of Stichodactyla helianthus

 
1.C.38.1.3Tenebrosin-A (fragment)AnimalsTenebrosin-A of Actinia tenebrosa (P30833)
 
1.C.38.1.4Actinoporin Or-A, cation-selective pore forming tetrameric toxin AnimalsActinoporin Or-A of Oulactis orientalis (sea anenome) (Q5I4B8)
 
1.C.38.1.5

Echotoxin-2 precursor, Echt-2 hemolysin (276 aas). Pore-forming protein; forms cation-selective hydrophilic pores of around 1 nm and causes cardiac stimulation and hemolysis. Pore formation is a multi-step process that involves recognition of membrane sphingomyelin using aromatic rich regions and adjacent phosphocholine binding sites for firm binding to the membrane accompanied by the transfer of the N-terminal region to the lipid-water interface and finally pore formation after oligomerization of several monomers (Kawashima et al., 2003; Shiomi et al., 2002).

Animals

Echt-2 hemolysin of Monplex echo (a marine gastropod)
(Q76CA2)

 
1.C.38.1.6Cytolytic pore-forming tetrameric toxin (forms cation-selective pores (d = 1 nm) (Mebs et al., 1992). AnimalsCytolysin of Heteractis magnifica
(P39088)
 
1.C.38.1.7The plant actinoporin homologue (293aas). Function unknown.

Plants

Actinoporin homologue of Physcomitrella patens (A9S8W4)

 
1.C.38.1.8

Fragaceatoxin C (FraC) of the strawberry anemone (Structure solved to 1.8 Å resolution (PPDB acc # 4TSL); It is a crown-shaped nonamer with an external diameter of about 11.0 nm and an internal diameter of approximately 5.0 nm.) Almost identical to Equinatoxin II/Tenebrosin C (1.C.38.1.1) (Mechaly et al., 2011).  Fragaceatoxin C (FraC) is an α-barrel pore-forming toxin (PFT). The crystal structures of FraC at four different stages of the lytic mechanism have been determined at 3.1Å resolution, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore (Tanaka et al. 2015). 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 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 this haemolytic toxin.  It has been reconstituted in  planar lipid bilayers and engineered for DNA analysis.  It shows a funnel-shaped geometry that allows tight wrapping around single-stranded DNA (ssDNA), resolving between homopolymeric C, T, and A polynucleotide stretches (Wloka et al. 2016). Despite the 1.2 nm internal constriction in the FraC pore, double-stranded DNA (dsDNA) can translocate through the nanopore at high applied potentials, presumably through deformation of the alpha-helical transmembrane region (Huang et al. 2017). Therefore, FraC nanopores might be useful for DNA sequencing and dsDNA analysis. Pore formation goes through a dimer intermediate and then generates the active octamer. Disrupting the key hydrophobic interaction between V60 and F163 (FraC numbering scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII) and sticholysin II (StII) impairs the pore formation activity (Mesa-Galloso et al. 2016). It was reviewed by Gupta et al. 2023. FraE is almot identical to this protein.

Animals

FraC of Actine fragacea (B9W5G6)

 
1.C.38.1.9

Equinatoxin 5 of 214 aas (Frazão et al. 2012). Bryoporin-7 (P61914; 214 aas) is 83% identical.

Animals

Equinatoxin-5 of Actinia equina
 
Examples:

TC#NameOrganismal TypeExample
Examples:

TC#NameOrganismal TypeExample