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Abstract Sticholysin I and II (Sts: St I and St II) are proteins of biomedical interest that form pores upon the insertion of their N-terminus in the plasma membrane. Peptides spanning the N-terminal residues of StI (StI 1-31) or StII (StII 1-30) can mimic the permeabilizing ability of these toxins, emerging as candidates to rationalize their potential biomedical applications. These peptides have different activities that correlate with their hydrophobicity. However, it is not clear how this property contributes to peptide folding in solution or upon binding to membranes. Here we compared the conformational properties of these peptides and shorter versions lacking the hydrophobic segment 1–11 of StI (StI 12-31) or 1–10 of StII (StII 11-30). Folding of peptides was assessed in solution and in membrane mimetic systems and related with their ability to bind to membranes and to permeabilize lipid vesicles. Our results suggest that the differences in activity among peptides could be ascribed to their different folding propensity and different membrane binding properties. In solution, StII 1-30 tends to acquire α-helical conformation coexisting with self-associated structures, while StI 1-31 remains structureless. Both peptides fold as α-helix in membrane; but StII 1-30 also self-associates in the lipid environment, a process that is favored by its higher affinity for membrane. We stress the contribution of the non-polar/polar balance of the 1–10 amino acid sequence of the peptides as a determining factor for different self-association capabilities. Such difference in hydrophobicity seems to determine the molecular path of peptides folding upon binding to membranes, with an impact in their permeabilizing activity. This study contributes to a better understanding of the molecular mechanisms underlying the permeabilizing activity of Sts N-terminal derived peptides, with connotation for the exploitation of these small molecules as alternative of the full-length toxins in clinical settings. Graphical abstract Image 1 Highlights • Hydrophobicity of peptides StI 1-31 and StII 1-30 determines their self-association capabilities. • StII 1-30 but not StI 1-31 self-associates in solution and in the lipid environment. • Folding of StI 1-31 and StII 1-30 peptides is strongly conditioned by the membrane environment. • The higher hydrophobicity of StII 1-30 facilitates its partition into the membrane. • The permeabilizing activity of StII 1-30 could be favored by pre-association in solution. [ABSTRACT FROM AUTHOR]

A bstract Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that being secreted as soluble monomers are able to bind and permeabilize membranes leading to cell death. The interest in these proteins has risen due to their high cytotoxicity that can be properly used to design immunotoxins against tumor cells and antigen-releasing systems to cell cytosol. In this work we describe a novel actinoporin produced by Anthopleura nigrescens, an anemone found in the Central American Pacific Ocean. Here we report the amino acid sequence of an actinoporin as deduced from cDNA obtained from total body RNA. The synthetic DNA sequence encoding for one cytolysin variant was expressed in BL21 Star (DE3) Escherichia coli and the protein purified by chromatography on CM Sephadex C-25 with more than 97% homogeneity as verified by MS-MS and HPLC analyses. This actinoporin comprises 179 amino acid residues, consistent with its observed isotope-averaged molecular mass of 19 661 Da. 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% of α-helix, and 35% of random structure. Moreover, 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). Comparison by spectroscopic techniques and modeling the three-dimensional structure of nigrelysin (Ng) showed a high homology with StII but several differences were also detectable. Taken together, these results reinforce the notion that Ng is a novel member of the actinoporin pore-forming toxin (PFT) family with a HA as high as that of StII, the most potent actinoporin so far described, but with peculiar structural characteristics contributing to expand the understanding of the structure-function relationship in this protein family. Highlights • Nigrelysin (Ng) is a novel 20 kDa actinoporin produced by sea anemone A. nigrescens. • Ng efficiently permeabilizes cells and model membranes containing sphingomyelin. • Ng´s high permeabilizing activity is similar to that of StII with C 50 values ∼2 nM. [ABSTRACT FROM AUTHOR]

Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus that belongs to the actinoporin protein family. The high affinity of actinoporins for sphingomyelin (SM)-containing membranes has been well documented. However, the molecular determinants that define this affinity have not been fully clarified. Here, we have examined the binding and permeabilizing activity of StII to different single and mixed lipidic systems by combining lipid monolayers, liposomes, and permeabilizing assays. This study characterizes the contribution of ceramide-derived compounds for StII-membrane interaction. Molecular dynamics simulations revealed a differential binding mode of StII with the polar head group of SM and PC. The electrostatic interaction energies were the major energetic contributors to the better affinity of StII for SM compared to PC, while the van der Waals interaction energies were the major driving forces of the better affinity of StII for SM respect to Cer. Furthermore, the presence of sugar residues in glycosphingolipids modulated binding and pore-formation by actinoporins probably by hindering StII to reach relevant structural motifs in membrane for binding or inducing a non-competent adsorption to membrane. Our results demonstrate that StII-membrane interaction, leading to pore formation, may critically respond to changes in lipid head group properties, and the access to SM interfacial structural motif. [ABSTRACT FROM AUTHOR]

Actinoporins are pore-forming toxins (PFT) produced by sea anemones with molecular mass around 20 kDa and high affinity for sphingomyelin. The most studied atinoporins are sticholysins I and II (StI/StII) from Stichodactyla helianthus , equinatoxin II (EqtII) from Actinia equina , and fragaceatoxin C (FraC) from Actinia fragacea . Their N-terminal sequences encompassing residues 1–30 seem to be the best candidates for pore formation. This segment comprises an amphipathic α-helix preceded by a more or less hydrophobic segment, depending on the toxin, of around 10 amino acid residues. Although it is clear that the N-terminal is the most variable sequence in this protein family, the role of their hydrophobic segment in not fully understood. Here we show a comparison of StI, StII, EqtII, and FraC activities with that of their respective N-terminal synthetic peptides. The hemolytic and permeabilizing activity of the peptides reproduce qualitatively the behavior of their respective parental proteins and are particularly related to the hydrophobicity of the corresponding 1–10 segment. Furthermore, the dendrogram analysis of actinoporins' N-terminal sequence allows relating differences in alignment with differences in activity among the four toxins. We have also evaluated the penetration depth of the N-terminal segment of StI and StII by using Trp-containing peptide-analogs. Our data suggest that the N-terminus of StII is more deeply buried into the hydrophobic core of the bilayer than that of StI. We hypothesize that the highest activity of StII could be ascribed to a larger hydrophobic continuum , an uninterrupted sequence of non-charged mainly hydrophobic amino acid residues, of its N-terminus promoting a highest ability to partially insert in the membrane core. Moreover, as we show for four related peptides that a higher hydrophobicity contributes to increase the activity, we reinforce the notion that this property must be taken into account to design new potent membranotropic agents. [ABSTRACT FROM AUTHOR]