Avtor/Urednik		Hong, Qi; Gutierrez-Aguirres, Ion; Barlič, Ariana; Malovrh, Petra; Kristan, Katarina; Podlesek, Zdravko; Maček, Peter; Turk, Dušan; Gonzales-Manas, Juan M; Anderluh, Gregor
Naslov		Two-step membrane binding by equinatoxin II, a pore-forming toxin from the sea anemone, involves and exposed aromatic cluster and a flexible helix
Tip		članek
Vir		J Biol Chem
Vol. in št.		Letnik 277, št. 44
Leto izdaje		2002
Obseg		str. 41916-24
Jezik		eng
Abstrakt		Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3-4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membraneembedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.
Deskriptorji		CNIDARIAN VENOMS CYTOTOXINS PORINS LIPID BILAYERS BINDING SITES SEA ANEMONES AVIDIN HEMOLYSINS TRYPTOPHAN CYSTEINE