Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich

J Biol Chem. 2004 Nov 5;279(45):46509-17. doi: 10.1074/jbc.M406193200. Epub 2004 Aug 20.


Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp117. These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Circular Dichroism
  • Cloning, Molecular
  • Cnidarian Venoms / chemistry*
  • Cnidarian Venoms / pharmacology*
  • Crystallography, X-Ray
  • Cysteine / chemistry
  • Disulfides / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Erythrocytes / metabolism
  • Hemolysis
  • Humans
  • Lipids / chemistry
  • Liposomes / metabolism
  • Models, Molecular
  • Mutagenesis
  • Mutation
  • Oxidants / pharmacology
  • Oxygen / metabolism
  • Pressure
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Sea Anemones
  • Temperature
  • Time Factors
  • Tryptophan / chemistry


  • Cnidarian Venoms
  • Disulfides
  • Lipids
  • Liposomes
  • Oxidants
  • equinatoxin
  • Tryptophan
  • Cysteine
  • Oxygen

Associated data

  • PDB/1TZQ