title: Neutron reflection study of the interaction of the eukaryotic pore-forming actinoporin equinatoxin II with lipid membranes reveals intermediate states in pore formation.
abstract: Equinatoxin II (EqtII), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal α-helix into the membrane. EqtII 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 EqtII binding and insertion by using neutron reflection to determine the structures of EqtII-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 EqtII, 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 EqtII in which the α-helix is covalently tethered to the rest of the protein, interacts only reversibly with all the membranes. Releasing the α-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.
authors.affiliation: European Spallation Source ERIC, P.O. Box 176, 221 00 Lund, Sweden; Division of Physical Chemistry, Department of Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden; Institut Laue-Langevin, BP 156, 38042 Grenoble, France.
authors.forename: Hanna P
authors.initials: HP
authors.lastname: Wacklin
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia.
authors.forename: Biserka Bakrač
authors.initials: BB
authors.lastname: Bremec
authors.affiliation: Institut Laue-Langevin, BP 156, 38042 Grenoble, France.
authors.forename: Martina
authors.initials: M
authors.lastname: Moulin
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
authors.forename: Nejc
authors.initials: N
authors.lastname: Rojko
authors.affiliation: Institut Laue-Langevin, BP 156, 38042 Grenoble, France.
authors.forename: Michael
authors.initials: M
authors.lastname: Haertlein
authors.affiliation: Institut Laue-Langevin, BP 156, 38042 Grenoble, France.
authors.forename: Trevor
authors.initials: T
authors.lastname: Forsyth
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia. Electronic address: gregor.anderluh@ki.si.
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia. Electronic address: ray.norton@monash.edu.
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Norton
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D003064:Cnidarian Venoms / Q000737:chemistry / Q000633:toxicity; D004134:Dimyristoylphosphatidylcholine; D008051:Lipid Bilayers / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D009502:Neutrons; D054353:Perforin / Q000737:chemistry* / Q000378:metabolism; D044367:Phase Transition; D016062:Porosity; D017433:Protein Structure, Secondary; D012615:Sea Anemones / Q000737:chemistry; D013109:Sphingomyelins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003064:Cnidarian Venoms; D008051:Lipid Bilayers; D008055:Lipids; D008563:Membrane Lipids; D013109:Sphingomyelins; D054353:Perforin; C012307:equinatoxin; D002784:Cholesterol; D004134:Dimyristoylphosphatidylcholine
keywords: Cholesterol; Cytolysin; Deuteration; Equinatoxin II; Membrane; Neutron reflection; Pore formation; Sphingomyelin