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Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2004 Nov 05; Vol. 279 (45), pp. 46509-17. Date of Electronic Publication: 2004 Aug 20. - Publication Year :
- 2004
-
Abstract
- 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.
- Subjects :
- Animals
Blotting, Western
Circular Dichroism
Cloning, Molecular
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 chemistry
Cnidarian Venoms pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 279
- Issue :
- 45
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15322132
- Full Text :
- https://doi.org/10.1074/jbc.M406193200