Molecular Basis of the Sticholysin-Membrane Interaction: On the Structure of the Pore and the Effects of Lipids

The usefulness of toxicity across the tree of life is far beyond doubt. Most, if not all, organisms produce compounds that can be used for attack and/or defense against external entities. Some of the most specialized of these compounds are toxic proteins, among which pore-forming toxins (PFTs) particularly excel. PFTs are present in all kingdoms of life. Given the wide variety of PFTs, one can expect a multitude of different specificities and mechanisms of action, of which we will certainly take advantage at some point. For that, a thorough characterization of PFTs and their functionality is necessary. In this thesis, we have taken further the characterization of sticholysins, small PFTs produced by the sea anemone Stichodactyla helianthus.

In paper I, the influence of bilayer thickness on the pore forming activity of sticholysins has been studied. Model lipid bilayers made mainly of a phosphatidylcholine (PC), with two monounsaturated acyl chains that determined membrane thickness, were used. Myristoyl-sphingomyelin (14:0-SM) was included to ensure membrane recognition by sticholysins. The effect of cholesterol (Chol) was also evaluated. The preferred thickness for sticholysins was that of di-18:1-PC membranes. This seems to be a result of evolutionary pressure since it agrees with the most common acyl chains found in analyzed fish.

In paper II, different fluorescent derivatized lipids, Sticholysin II (StnII) tryptophan mutants, and oleoyl-ceramide (OCer) were used to delve into the relationship between StnII and Chol and sphingomyelin (SM). We found that Chol favored SM recognition by StnII while, concomitantly, StnII rearranged the membrane, extracting Chol from the SM-rich domains. In fact, Chol was preferentially distributed near StnII.

In paper III, we evaluated the mechanism of the release of aqueous contents from vesicles. Several different probes were employed. We found that the StnII pore is too small for calcein to leak through. We propose that molecules of comparable size to calcein are released through the membrane perturbations produced by StnII during pore formation. The final pore would only let through very small molecules, such as dithionite.

In paper IV, a single-cysteine mutant of StnI was used to study oligomerization directly in unrestrained environments, such as that of model membranes. The results are consistent with previous structures obtained for other actinoporins. Furthermore, we have observed that the stoichiometry of the complex was maintained when StnII was included in the complex. The results in solution also support the previously observed ability of StnII to promote StnI binding to membranes.