Functional characterization of Val60, a key residue involved in the membrane‐oligomerization of fragaceatoxin C, an actinoporin from <i>Actinia fragacea</i>

Morante, Koldo; Caaveiro, José M. M.; Viguera, Ana Rosa; Tsumoto, Kouhei; González-Mañas, Juan Manuel

doi:10.1016/j.febslet.2015.06.012

Cited by 20 publications

(21 citation statements)

References 30 publications

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“…Many of the actinoporin candidates in clade 2A are instead equipped with KGD (R144K) residues, and the actinoporin candidates in clade 2M are equipped with EGD (R144E) residues or have a deletion in this region (Figure 2). Additionally, other residues thought to play a key role in oligomerization (I59 and V60) were highly variable [25] and did not appear to follow any phylogenetic pattern (Figure 2). …”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Several residues have been manipulated to identify functionally important regions within the protein [25], revealing an aromatic-rich region that forms the phosphocholine (POC) binding site, with a single amino acid residue (W112 in Equinatoxin II (EqII)) taking on a key role in initiating sphingomyelin recognition and pore formation [11,26,27]. Although events leading to oligomerization remain uncertain, both the RGD domain (R144, G145, and D146 in EqII) and a single valine residue (V60 in EqII) are thought to direct protein attachment and play a key role in this process [25,28]. Ultimately, a key hydrophobic arginine (R31 in EqII) and other hydrophobic residues in the α-helix at the N-terminal region are involved in cell membrane penetration and the formation of the ion conductive pathway [29,30,31,32,33,34], forming a selective pore from four monomers [12,35,36], although oligomerizations involving eight or nine peptides have also been proposed [37,38].…”

Section: Introductionmentioning

confidence: 99%

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Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones

Macrander

Daly

2016

Toxins

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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. We used a combined bioinformatic and phylogenetic approach to investigate how actinoporins have evolved across three superfamilies of sea anemones (Actinioidea, Metridioidea, and Actinostoloidea). Our analysis identified 90 candidate actinoporins across 20 species. We 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. Although multiple residues are involved in initiating sphingomyelin recognition and membrane binding, there is a high rate of replacement for a specific tryptophan with leucine (W112L) and other hydrophobic residues. 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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones

Macrander

Daly

2016

Toxins

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“…The latest actinoporin pore-like oligomeric structure published is a crystalline octameric ensemble of FraC (66). According to those results, the two side-chain residues showing more buried surface upon oligomerization would be FraC Val-60 and Trp-149 (66,80). These residues have their corresponding counterparts in StnI (Ile-59 and Trp-149) and StnII (Ile-58 and Trp-146).…”

Section: 77-79)mentioning

confidence: 94%

Synergistic Action of Actinoporin Isoforms from the Same Sea Anemone Species Assembled into Functionally Active Heteropores

Rivera-de-Torre

García‐Linares

Alegre-Cebollada

et al. 2016

Journal of Biological Chemistry

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Among the toxic polypeptides secreted in the venom of sea anemones, actinoporins are the pore-forming toxins whose toxic activity relies on the formation of oligomeric pores within biological membranes. Intriguingly, actinoporins appear as multigene families that give rise to many protein isoforms in the same individual displaying high sequence identities but large functional differences. However, the evolutionary advantage of producing such similar isotoxins is not fully understood. Here, using sticholysins I and II (StnI and StnII) from the sea anemone Stichodactyla helianthus, it is shown that actinoporin isoforms can potentiate each other's activity. Through hemolysis and calcein releasing assays, it is revealed that mixtures of StnI and StnII are more lytic than equivalent preparations of the corresponding isolated isoforms. It is then proposed that this synergy is due to the assembly of heteropores because (i) StnI and StnII can be chemically cross-linked at the membrane and (ii) the affinity of sticholysin mixtures for the membrane is increased with respect to any of them acting in isolation, as revealed by isothermal titration calorimetry experiments. These results help us understand the multigene nature of actinoporins and may be extended to other families of toxins that require oligomerization to exert toxicity.Actinoporins, single polypeptide chains of around 175 amino acids, constitute a family of toxic proteins produced by different sea anemone species. They show basic isoelectric point values and are usually cysteineless (1-4). Actinoporins belong to a much larger group of widely distributed proteins, known as pore-forming toxins, whose toxic activity relies on the formation of pores within biological membranes (5-9). All poreforming toxins show a very similar dual behavior by which they remain mostly monomeric and stably folded in aqueous solution but become oligomeric integral proteins when encountering membranes (2-4, 10 -23).The incorporation of actinoporins into the membrane largely depends on lipid bilayer composition and membrane physicochemical state (18, 24 -29). Both factors influence the conformational changes occurring during the transition from the water media to the inserted states of the protein (30, 31). Thus, high affinity recognition of sphingomyelin (SM) 3 is crucial for specific attachment to a membrane, but the subsequent effects observed also depend on the physical properties derived from its particular composition and not only from its SM content (23, 32). In fact, although still controversial, the presence of cholesterol and the coexistence of different phases in the membrane seem to be important factors, if not for binding then at least for the final formation of the pore (23,28,29,(32)(33)(34)(35)(36).Actinoporins have been isolated from more than 20 different sea anemone species (1, 3, 37-41) in agreement with their rather ubiquitous distribution within the Actinaria order (1). They display high sequence identities (between 60 and 80%) and appear as multigene families, ...

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“…Expression and purification of FraC were carried out as described previously (22). Briefly, FraC expression was induced in E. coli BL21 (DE3) cells and then purified to homogeneity by ion-exchange and size-exclusion chromatography.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins

Morante

Bellomio

Gil

et al. 2016

Journal of Biological Chemistry

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Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. 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 known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C 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. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an α-PFT.

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Functional characterization of Val60, a key residue involved in the membrane‐oligomerization of fragaceatoxin C, an actinoporin from Actinia fragacea

Cited by 20 publications

References 30 publications

Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones

Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones

Synergistic Action of Actinoporin Isoforms from the Same Sea Anemone Species Assembled into Functionally Active Heteropores

Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins

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