Neutron reflection study of the interaction of the eukaryotic pore-forming actinoporin equinatoxin II with lipid membranes reveals intermediate states in pore formation

Equinatoxin II (Eqtll), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal alpha-helix into the membrane. Eqtll pore formation is dependent on sphingomyelin (SM), although cholesterol (Chol) and membrane microdomains have also been suggested to enhance its activity. We have investigated the mechanism of Eqtll binding and insertion by using neutron reflection to determine the structures of Eqtll-membrane assemblies in situ. EqtII has several different modes of binding to membranes depending on the lipid composition. In pure dimyristoyl-phosphatidylcholine (DMPC) membranes, EqtII interacts weakly and reversibly with the lipid head groups in an orientation approximately parallel to the membrane surface. The presence of sphingomyelin (SM) gives rise to a more upright orientation of Eqtll, but Chol is required for insertion into the core of the membrane. Cooling the EqtII-lipid assembly below the lipid phase transition temperature leads to deep water penetration and a significant reduction in the extension of the protein outside the membrane, indicating that phase-separation plays a role in EqtII pore-formation. An inactive double-cysteine mutant of Eqtll in which the alpha-helix is covalently tethered to the rest of the protein, interacts only reversibly with all the membranes. Releasing the alpha-helix in situ by reduction of the disulphide bridge, however, causes the mutant protein to penetrate in DMPC-SM-Chol membranes in a manner identical to that of the wild-type protein. Our results help clarify the early steps in pore formation by EqtII and highlight the valuable information on protein-membrane interactions available from neutron reflection measurements. (C) 2015 Elsevier B.V. All rights reserved.