Author/Editor		Kristan, Katarina; Podlesek, Zdravko; Hojnik, Vesna; Gutierrez-Aguirre, Ion; Gunčar, Gregor; Turk, Dušan; Gonzales-Manas, Juan M; Lakey, Jeremy H; Maček, Peter; Anderluh, Gregor
Title		Pore formation by equinqtoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich
Type		članek
Source		J Biol Chem
Vol. and No.		Letnik 279, št. 45
Publication year		2004
Volume		str. 46509-17
Language		eng
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 (3-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal a-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.
Descriptors		SEA ANEMONES CNIDARIAN VENOMS PORINS MUTATION BINDING SITES CYSTEINE CLONING, MOLECULAR HEMOLYSINS DISULFIDES ESCHERICHIA COLI