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J. Biol. Chem., Vol. 277, Issue 7, 5351-5359, February 15, 2002

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A Mutational Analysis of the Globotriaosylceramide-binding Sites of Verotoxin VT1^*

Anna M. Soltyk^§, C. Roger MacKenzie^¶, Vince M. Wolski^§, Tomoko Hirama^¶, Pavel I. Kitov, David R. Bundle, and James L. Brunton^§**

From the  Clinical Science Division, University of Toronto, Toronto, Ontario M5S 1A8, Canada, the ^§ Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, the ^¶ Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada, the  Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada, and the ^** Toronto General Hospital, Toronto, Ontario M5G 2C4, Canada

Escherichia coli verotoxin, also known as Shiga-like toxin, binds to eukaryotic cell membranes via the glycolipid Gb₃ receptors which present the P^k trisaccharide Gal(14)Gal(1-4)Glc. Crystallographic studies have identified three P^k trisaccharide (P^k-glycoside) binding sites per verotoxin 1B subunit (VT1B) monomer while NMR studies have identified binding of P^k-glycoside only at site 2. To understand the basis for this difference, we studied binding of wild type VT1B and VT1B mutants, defective at one or more of the three sites, to P^k-glycoside and pentavalent P^k trisaccharide (pentaSTARFISH) in solution and Gb₃ presented on liposomal membranes using surface plasmon resonance. Site 2 was the key site in terms of free trisaccharide binding since mutants altered at sites 1 and 3 bound this ligand with wild type affinity. However, effective binding of the pentaSTARFISH molecule also required a functional site 3, suggesting that site 3 promotes pentavalent binding of linked trisaccharides at site 1 and site 2. Optimal binding to membrane-associated Gb₃ involved all three sites. Binding of all single site mutants to liposomal Gb₃ was weaker than wild type VT1B binding. Site 3 mutants behaved as if they had reduced ability to enter into high avidity interactions with Gb₃ in the membrane context. Double mutants at site 1/site 3 and site 2/site 3 were completely inactive in terms of binding to liposomal Gb_3, even though the site 1/site 3 mutant bound trisaccharide with almost wild type affinity. Thus site 2 alone is not sufficient to confer high avidity binding to membrane-localized Gb₃. Cytotoxic activity paralleled membrane glycolipid binding. Our data show that the interaction of verotoxin with the Gb₃ trisaccharide is highly context dependent and that a membrane environment is required for biologically relevant studies of the interaction.

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