Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Abstract

Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp 112 and Tyr 113 exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins. Sphingomyelin (SM)8 is an important eukaryotic membrane lipid, located for the most part in the outer leaflet of the plasma membrane in the form of specialized cholesterol-rich microdomains, so-called lipid rafts (1, 2). Many pathogens and toxic proteins employ lipid rafts to invade cells (3, 4), but currently little is known about the molecular details of the recognition mechanism of the lipid components present in the rafts. In the particular case of SM, the specific recognition occurs even though SM exposes the same phosphorylcholine headgroup as the other abundant lipid, phosphatidylcholine. SM-binding proteins are currently exploited as specific markers for cellular SM (5) and are used to identify other proteins involved in sphingolipid metabolism (6).Actinoporins are extremely potent cytolysins produced exclusively by sea anemones (7,8). They may be used to capture prey, in intraspecific aggression, or in preventing adhesion of other organisms (7, 9). The two most studied representatives are EqtII, isolated from the sea anemone Actinia equina, and sticholysin II (StII) from Stichodactyla helianthus. Actinoporins constitute a family of conserved proteins that cause hemolysis of red blood cells by colloid-osmotic lysis and exhibit cytolytic activity against various cell lines (7, 10 -12). Even the most distant members of the family share more than 60% sequence identity and the available threedimensional structures of EqtII and StII are nearly superimposable (13-15). The structure is composed of a tightly folded ␤-sandwich flanked b...

Membrane Damage by an α-Helical Pore-forming Protein, Equinatoxin II, Proceeds through a Succession of Ordered Steps

“…The labeling at position 179 has been shown not to affect permeabilizing activity (35), and it is fully exposed to the solvent when the protein is in solution or bound to membranes (17,35). The C terminus is located on the side opposite that occupied by regions of the molecule that participate in membrane binding (18,19,36) or formation of the final pore (23,25), and any labeling does not interfere with various steps in pore formation. This position would thus be suitable to report oligomerization at the surface of the membrane, when labeled with convenient fluorescent probes.…”

Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins, pore-forming proteins from a sea anemone

“…However, the exact sequence of events that takes place during the assembly of actinoporins and the identification of the functionally relevant intermediates in the membrane remain under debate. It is now generally accepted that actinoporins bind to membranes mainly as monomers (Tejuca et al 1996;Barlic et al 2004;Bakrac et al 2008;Pedrera et al 2014;Rojko et al 2014) and then undergo a conformational change that involves only the N-terminal segment (Rojko et al 2013(Rojko et al , 2015Tanaka et al 2015). The monomeric units then oligomerize to form pores in which the N-terminal α-helix lines the channel walls in conjunction with lipids (Tanaka et al 2015).…”

“…Co-crystallization of StII with POC enabled the definition of the POC binding site, crucial for the specific recognition of SM. Residues involved in the POC binding site are strictly conserved in actinoporins, implying that the same mechanism of lipid headgroup recognition is followed by other members of this protein family (Bakrac et al 2008). The POC binding site is a cavity formed by amino acids of the second α-helix, the compact β-sheet nucleus and the aromatic amino acid cluster (Fig.…”

“…1). This binding site together with the aromatic amino acid cluster forms an essential structural assembly for binding of these toxins to membranes and is known as the interfacial binding site (Bakrac et al 2008).…”

Ostreolysin A/Pleurotolysin B and Equinatoxins: Structure, Function and Pathophysiological Effects of These Pore-Forming Proteins

“…EqT II recognises the sphingomyelin head group [5], while Oly specifically binds to membrane domains containing both sphingomyelin and cholesterol [2,6]. This binding results in protein oligomerization at the membrane surface, and the formation of transmembrane pores, leading to colloid-osmotic mechanisms resulting in cell lysis.…”