Sticholysin II (StII) is a water soluble protein produced by the Caribbean sea anemone Stichodactyla heliantus that binds to membranes, causing lysis through pore formation. Due to StII affinity for sphingomyelin (SM), this lipid has been postulated as its membrane receptor. However, the toxin mechanism of action is not fully understood at the molecular level. Structure resolution by means of x-ray crystallography showed that the protein is formed by a rigid body of b sheets flanked by two small a-helices, one of which is an amphipathic a-helix (residues 14 - 24) located in the N-terminal region. Pore formation requires oligomerization of three to four monomers at the membrane interface. StII´s N-terminal region has been proposed to have an important role in pore formation. To examine this hypothesis, we studied the interaction between a peptide containing residues 1-30 (P1-30) of StII and small unilamelar vesicles (SUV), containing variable quantities of egg phosphatidylcholine  (ePC), SM, and negatively charged phosphatidic acid (PA) by means of circular dichroism (CD).

P1-30 was obtained by solid phase synthesis. Stock solutions were prepared in deionized water. Phospholipids were from Avanti Polar Lipids, Alabaster, Alabama. Lipid stock solutions were prepared in chloroform. The solvent was evaporated under a stream of nitrogen and the lipid film was suspended in aqueous solution. SUV were obtained by submitting the lipid dispersions to ultrasonic radiation (Branson 450 Sonifier) under a N₂ atmosphere in an ice bath, for 5 min and 1 min pause at medium power. This process was repeated until the solutions became transparent. Possible titanium contamination (from the sonifier tip) was removed by centrifugation in a Micro 22R Hettich centrifuge. Final phospholipid concentrations in the SUV and in the stock chloroform solutions were measured according to the method of Rouser et al (Lipids, 5 494-496, 1970) for phosphate determination. CD spectra were obtained with a Jasco 720 spectropolarimeter, in the far UV region (195 – 260 nm). Quartz cells of 5.0 mm optical path were used.

P1-30 aggregates in aqueous solution (pH 7) in the absence of membranes, probably due to the highly hydrophobic segment in residues 1-10. Upon binding to membranes, disaggregation occurred concomitantly with an increase of secondary structure content. The peptide acquired a-helical conformation that increased with the increase of the SUV/peptide ratio. Addition of PA to ePC bilayers enhanced binding and caused an increase of a-helical conformation, very likely due to electrostatic interactions between the positively charged peptide and the negatively charged membrane. Moreover, it is also possible that peptide binding is favored by PA-promoted negative curvature.  When SM was added as the third component the binding of P1-30 increased. As a result, the peptide a-helical content also increased. The SM-induced enhanced binding could be due to phase separation in the SM-ePC-PA system, since it is known that SM tends to form a more organized phase than ePC and PA. This phenomenon could cause membrane defects and, thereby, facilitate insertion of the peptide hydrophobic region in the bilayer.

The results clearly point to the importance of lipid composition, in particular, the presence of SM, for the interaction between StII N-terminus and model membranes. Moreover, the CD spectra indicate that binding of the peptide to the model membranes is coupled to the acquisition of a-helical conformation. Insertion of the toxin N-terminal region in the membrane and acquisition of helical structure could represent the initial steps of pore formation.