title: A novel mechanism of pore formation: membrane penetration by the N-terminal amphipathic region of equinatoxin.
abstract: Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10-28 are organized as an alpha-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation.
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Petra
authors.initials: P
authors.lastname: Malovrh
authors.forename: Gabriella
authors.initials: G
authors.lastname: Viero
authors.forename: Mauro Dalla
authors.initials: MD
authors.lastname: Serra
authors.forename: Zdravko
authors.initials: Z
authors.lastname: Podlesek
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Lakey
authors.forename: Peter
authors.initials: P
authors.lastname: Macek
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Menestrina
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular; D003064:Cnidarian Venoms / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics*; D005456:Fluorescent Dyes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000493:pharmacokinetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000493:pharmacokinetics; D012615:Sea Anemones
chemical_list: D003064:Cnidarian Venoms; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C012307:equinatoxin