Sticholysin II (StnII) is a pore-forming protein (PFP) produced by the sea anemone Stichodactyla helianthus. We found out that StnII exists in a monomeric soluble state but forms tetramers in the presence of a lipidic interface. Both structures have been independently determined at 1.7 A and 18 A resolution, respectively, by using X-ray crystallography and electron microscopy of two-dimensional crystals. Besides, the structure of soluble StnII complexed with phosphocholine, determined at 2.4 A resolution, reveals a phospholipid headgroup binding site, which is located in a region with an unusually high abundance of aromatic residues. Fitting of the atomic model into the electron microscopy density envelope suggests that while the beta sandwich structure of the protein remains intact upon oligomerization, the N-terminal region and a flexible and highly basic loop undergo significant conformational changes. These results provide the structural basis for the membrane recognition step of actinoporins and unexpected insights into the oligomerization step.

RNase T1 is the best known representative of a large family of ribonucleolytic proteins secreted by fungi, mostly Aspergillus and Penicillium species. Ribotoxins stand out among them by their cytotoxic character. They exert their toxic action by first entering the cells and then cleaving a single phosphodiester bond located within a universally conserved sequence of the large rRNA gene, known as the sarcin-ricin loop. This cleavage leads to inhibition of protein biosynthesis, followed by cellular death by apoptosis. Although no protein receptor has been found for ribotoxins, they preferentially kill cells showing altered membrane permeability, such as those that are infected with virus or transformed. Many steps of the cytotoxic process have been elucidated at the molecular level by means of a variety of methodological approaches and the construction and purification of different mutant versions of these ribotoxins. Ribotoxins have been used for the construction of immunotoxins, because of their cytotoxicity. Besides this activity, Aspf1, a ribotoxin produced by Aspergillus fumigatus, has been shown to be one of the major allergens involved in allergic aspergillosis-related pathologies. Protein engineering and peptide synthesis have been used in order to understand the basis of these pathogenic mechanisms as well as to produce hypoallergenic proteins with potential diagnostic and immunotherapeutic applications.

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The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid-protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporins family, have been produced and their hemolytic and lipidbinding properties compared to those of the wild-type protein. A thermodynamical approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpydriven process that occurs with a high affinity constant (1.7 x 10 8 M -1 ). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, even though it is necessary for correct pore 2 formation. Membrane binding is achieved through two separate mechanisms, i.e.recognition of the lipid-water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site.Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation.3

A potent hemolytic polypeptide, sticholysin II, has been purified to homogeneity from the sea anemone Stichodactyla helianthus. The protein produces leakage of aqueous contents of model lipid vesicles composed of either phosphatidylcholine or sphingomyelin if cholesterol is present in these membranes. The leakage has been analyzed by measuring the dequenching of the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonic acid, coencapsulated with its quencher N,N′-p-xylenebispyridinium bromide, upon dilution of the vesicle contents into the external medium. The protein displays a maximum effect on vesicles containing 20Ϫ25% cholesterol. Leakage is also produced in vesicles composed of mixtures of phosphatidylcholine and sphingomyelin, the maximum effect being observed for 20Ϫ30% sphingomyelin molar content. The extent of the leakage is dependent on the molecular mass of the vesicle entrapped solutes in the range 445Ϫ960 Da. This suggests the involvement of a pore of about 1 nm in diameter based on the limiting size observed for the leakage of the different solutes. Oligomerization of the protein is apparently involved in the membrane permeabilization, based on the kinetic analysis of the leakage process which is shown to proceed through an all-or-none mechanism.Keywords : cytolysin; lipid vesicle; membrane permeabilization; protein-lipid interaction.Stichodactyla helianthus is a sea anemone occurring in the further isolated and resolved into two cytolytic proteins, sticholysin I and II, by ion-exchange chromatography (Díaz et al., coastal waters of the Caribbean region. Like other coelenterates, it produces many toxic peptides and proteins located within in-1992; Tejuca et al., 1996), that display very different primary structure. In contrast, it has been reported that two distinct anemtracellular specialized organelles called nematocysts and employed on the tentacles for defense and/or attack. These organ-one species produce a cytolytic protein of practically identical amino acid sequence. This is the case described for equinatoxin elles sting the prey by means of a rapid projection of a hollow tubule through which the toxic polypeptides are injected. The II from Actinia equina (Belmonte et al., 1994) and tenebrosin-C (Simpson et al., 1990) from Actinia tenebrosa. toxicity of these products, many of them are lethal substances, has been known for a long time. They can be divided into lowCytolysin III from S. helianthus, the most studied sea anemone toxin, was described as a potent hemolytic factor (Bernmolecular-mass (3Ϫ5 kDa) neurotoxins affecting sodium channels, and single polypeptide chain proteins (15Ϫ20 kDa), cyto-heimer and Avigad, 1976), although the extent of the effect was different depending on the origin of the mammalian erythrocytes lysins, acting on cell membranes (cytolytic effects towards red blood cells, platelets and fibroblasts have been reported ; Kem, considered. It was further demonstrated that the toxin increases the membrane permeability to small ions and solutes, probably 1988; Bernheimer...