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

Biserka Bakrač,Ion Gutiérrez-Aguirre,Zdravko Podlesek,Andreas F.-P Sonnen,Robert J.C Gilbert,Peter Maček,Jeremy H Lakey,Gregor Anderluh

doi:10.1074/jbc.m708747200

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.

Highlights

The equilibrium dissociation constants derived from the fits of sensorgrams presented in Fig. 6 are shown to the left, and critical pressures of lipid monolayer insertion derived from the plots in Fig. 7 are shown to the right for each lipid composition tested
We examined the ability of proteins to insert into lipid monolayers of the same lipid compositions used for SPR experiments
The results show that the natural variant W112L possesses wild-type lipid specificity. This mutant showed better association, insertion, and permeabilizing activity in membranes or monolayers composed of DOPC:POPG

Summary

Molecular Basis for Sphingomyelin Recognition

SM plays a major role in the binding of the toxin to the membrane (25). This was supported by the recent discovery of a phosphorylcholine (POC) binding site on the surface of StII (15). Lipid specificity has been poorly addressed in the available structural studies of actinoporins (13–15, 29, 30). It was shown by introducing a 19F label on EqtII tryptophans that Trp[112] participates in SM recognition, as it exhibits specific NMR chemical shift changes upon the addition of SM to PC micelles (31). In this work we have tested the hypothesis that EqtII can bind sphingomyelin both in solution and in lipid membranes. We show that the membrane-binding step is driven mainly by hydrophobic rather than electrostatic interactions and that the residues Trp[112] and Tyr[113] enable SM dependence

EXPERIMENTAL PROCEDURES

RESULTS

The electrostatic properties of

Summary of SPR and lipid monolayer results

DISCUSSION