A novel mechanism of pore formation: membrane penetration by the N-terminal amphipathic region of equinatoxin.
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.
Malovrh
Petra
P
Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia.
Viero
Gabriella
G
Serra
Mauro Dalla
MD
Podlesek
Zdravko
Z
Lakey
Jeremy H
JH
Macek
Peter
P
Menestrina
Gianfranco
G
Anderluh
Gregor
G
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
D003064:Cnidarian Venoms; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C012307:equinatoxin