Two-step membrane binding by Equinatoxin II, a pore-forming toxin from the sea anemone, involves an exposed aromatic cluster and a flexible helix.
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 membrane-embedded 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.
School of Biochemistry and Genetics, University of Newcastle upon Tyne, Framlington Place, United Kingdom.
Qi
Q
Hong
Ion
I
Gutierrez-Aguirre
Ariana
A
Barlic
Petra
P
Malovrh
Katarina
K
Kristan
Zdravko
Z
Podlesek
Peter
P
Macek
Dusan
D
Turk
Juan M
JM
Gonzalez-Manas
Jeremy H
JH
Lakey
Gregor
G
Anderluh
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000378:metabolism; D005453:Fluorescence; D008051:Lipid Bilayers / Q000378:metabolism; D017433:Protein Structure, Secondary; D055672:Static Electricity
D003064:Cnidarian Venoms; D008051:Lipid Bilayers; C012307:equinatoxin