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Two-step membrane binding by equinatoxin II, a pore-forming toxin from the sea anemone, involves an exposed aromatic cluster and a flexible helix

Lookup NU author(s): Dr Qi Hong, Professor Jeremy Lakey

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Abstract

Equinatoxin II (EqtlI) 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.


Publication metadata

Author(s): Hong Q, Gutierrez-Aguirre I, Barli A, Malovrh P, Kristan K, Podlesek Z, Maek P, Turk D, Gonzalez-Manas JM, Lakey JH, Anderluh G

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2002

Volume: 277

Issue: 44

Pages: 41916-41924

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

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

URL: http://dx.doi.org/10.1074/jbc.M204625200

DOI: 10.1074/jbc.M204625200

PubMed id: 12198118


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