Regulation of Sticholysin II-Induced Pore Formation by Lipid Bilayer Composition, Phase State, and Interfacial Properties

Abstract

Sticholysin II (StnII) is a pore-forming toxin that uses sphingomyelin (SM) as the recognition molecule in targeting membranes. After StnII monomers bind to SM, several toxin monomers act in concert to oligomerize into a functional pore. The regulation of StnII binding to SM, and the subsequent pore-formation process, is not fully understood. In this study, we examined how the biophysical properties of bilayers, originating from variations in the SM structure, from the presence of sterol species, or from the presence of increasingly polyunsaturated glycerophospholipids, affected StnII-induced pore formation. StnII-induced pore formation, as determined from calcein permeabilization, was fastest in the pure unsaturated SM bilayers. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/saturated SM bilayers (4:1 molar ratio), pore formation became slower as the chain length of the saturated SMs increased from 14 up to 24 carbons. In the POPC/palmitoyl-SM (16:0-SM) 4:1 bilayers, SM could not support pore formation by StnII if dimyristoyl-PC was included at 1:1 stoichiometry with 16:0-SM, suggesting that free clusters of SM were required for toxin binding and/or pore formation. Cholesterol and other sterols facilitated StnII-induced pore formation markedly, but the efficiency did not appear to correlate with the sterol structure. Benzyl alcohol was more efficient than sterols in enhancing the pore-formation process, suggesting that the effect on pore formation originated from alcohol-induced alteration of the hydrogen-bonding network in the SM-containing bilayers. Finally, we observed that pore formation by StnII was enhanced in the PC/16:0-SM 4:1 bilayers, in which the PC was increasingly unsaturated. We conclude that the physical state of bilayer lipids greatly affected pore formation by StnII. Phase boundaries were not required for pore formation, although SM in a gel state attenuated pore formation.

Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins

“…In these experiments, the fluorescent dye conjugated to the toxin colocalized with the DOPC domains ( dark areas in Fig. 2 C ) indicating that the toxin preferentially binds to the fluid phase domains than to the gel domains, a result consistent with the observations made with monolayers of FraC and leakage assays of sticholysin II (40). Based on these results, membranes composed of DOPC were selected for structural studies analyzing the conformation of FraC bound to membranes in a non-lytic scenario.…”

Functional and Structural Variation among Sticholysins, Pore-Forming Proteins from the Sea Anemone Stichodactyla helianthus

“…PFPs present different mechanisms to target their objective. The most common ones are the interaction of a specific lipid, like cholesterol (Chol) [53,[60][61][62][63][64][65][66][67][68][69][70][71][72][73] or sphingomyelin (SM) [12,25,26,44,69,[74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91], or recognition of a specific membrane protein receptor [32,[92][93][94][95][96]. It is common to find collections of highly prey-specific toxins as part of multigene families resulting in an extended range of different targets [97].…”

Pore-Forming Proteins from Cnidarians and Arachnids as Potential Biotechnological Tools

“…It has been largely accepted that actinoporins do not need a protein receptor to exert their toxicity, but instead require sphingomyelin as a specific lipidic receptor [16,17,26,27,28]. Furthermore, cholesterol, though not indispensable, plays a key role in their pore-forming mechanism [14,29,30,31,32,33,34], a mechanism still not fully understood, especially with regard to the sequence of events during pore formation and the final stoichiometry of the pore [35,36,37,38,39,40,41,42]. Overall, actinoporins represent a simple and optimal model to study the challenging biophysical transition from a water-soluble conformation to an integral transmembrane state.…”

“…However, actinoporins are very well studied from the structure-function point of view, allowing protein engineering to overcome these hurdles. Several research studies delve into the details of the protein-lipid interactions [10,15,30,31,33,42,99,100,101,102,103], making possible the application of different strategies directed to improve toxin specificity: for example, protecting the toxin region responsible for membrane binding or blocking the N-terminal domain directly implicated in pore formation. Actinoporins can be engineered to protect their key regions with polypeptide domains, which can later be released by tumor specific proteases.…”