Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich.
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.
Kristan
Katarina
K
Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia.
Podlesek
Zdravko
Z
Hojnik
Vesna
V
Gutiérrez-Aguirre
Ion
I
Guncar
Gregor
G
Turk
Dusan
D
González-Mañas
Juan M
JM
Lakey
Jeremy H
JH
Macek
Peter
P
Anderluh
Gregor
G
D000818:Animals; D015153:Blotting, Western; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D011312:Pressure; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012615:Sea Anemones; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D003064:Cnidarian Venoms; D004220:Disulfides; D008055:Lipids; D008081:Liposomes; D016877:Oxidants; C012307:equinatoxin; D014364:Tryptophan; D003545:Cysteine; D010100:Oxygen