Abstract
Actinoporins sticholysin I and sticholysin II (St I, St II) are proposed to lyse model and biomembranes via toroidal pore formation by their N-terminal domain. Based on the hypothesis that peptide fragments can reproduce the structure and function of this domain, the behavior of peptides containing St I residues 12–31 (StI12-31), St II residues 11–30 (StII11-30), and its TOAC-labeled analogue (N-TOAC-StII11-30) was examined. Molecular modeling showed a good match with experimental structures, indicating amphipathic α-helices in the same regions as in the toxins. CD spectra revealed that the peptides were essentially unstructured in aqueous solution, acquiring α-helical conformation upon interaction with micelles and large unilamellar vesicles (LUV) of variable lipid composition. Fluorescence quenching studies with NBD-containing lipids indicated that N-TOAC-StII11-30’s nitroxide moiety is located in the membranes polar head group region. Pyrene-labeled phospholipid inter-leaflet redistribution suggested that the peptides form toroidal pores, according to the mechanism of action proposed for the toxins. Binding occurred only to negatively charged LUV, indicating the importance of electrostatic interactions; in contrast the peptides bound to both negatively charged and zwitterionic micelles, pointing to a lesser influence of these interactions. In addition, differences between bilayers and micelles in head group packing and in curvature led to differences in peptide-membrane interaction. We propose that the peptides topography in micelles resembles that of the toxins in the toroidal pore. The peptides mimicked the toxins permeabilizing activity, St II peptides being more effective than StI12-31. To our knowledge, this is the first demonstration that differences in the toxins N-terminal amphipathic α-helix play a role in the difference between St I and St II activities.
Highlights
Sticholysin I and sticholysin II (St I and St II), cytolysins produced by the sea anemone Stichodactyla helianthus, belong to the family of actinoporins [1, 2]
To gain further insight into the molecular mechanism of pore formation, we report the synthesis of peptides comprising the N-terminal amphipathic α-helical segments of St I and St II
Several approaches were employed to study peptides corresponding to the N-terminal amphipathic α-helices of the sea anemone actinoporins sticholysin I and sticholysin II
Summary
Sticholysin I and sticholysin II (St I and St II), cytolysins produced by the sea anemone Stichodactyla helianthus, belong to the family of actinoporins [1, 2] These toxins present 93% sequence homology and act by forming pores in membranes, their putative receptor being the phospholipid sphingomyelin. High resolution solution studies—X-ray crystallography of St II [4] and NMR of St I [5]— indicated that the proteins are folded as a β-sandwich flanked by two α-helical segments, one in the N-terminal region and the other in the middle of the polypeptide chain In both cases the first 13 residues are essentially unordered, except for residues 4–8 of St II, which are in βsheet conformation. Equinatoxin II (Eqt II) [9, 10] and fragaceatoxin (FragC) [11] present similar threedimensional folds
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