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

Lookup NU author(s): Professor Jeremy LakeyORCiD


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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 β-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the β-sandwich and importantly the C-terminal α-helix, do not undergo large conformational rearrangements during the pore formation. The role of the β-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 β-sandwich are pre-requisite for proper pore formation by the actinoporin family.

Publication metadata

Author(s): Kristan K, Podlesek Z, Hojnik V, Gutierrez-Aguirre I, Guncar G, Turk D, Gonzalez-Manas JM, Lakey JH, Macek P, Anderluh G

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2004

Volume: 279

Issue: 45

Pages: 46509-46517

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology, Inc.


DOI: 10.1074/jbc.M406193200

PubMed id: 15322132


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