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Phenylalanine 30 plays an important role in receptor binding of verotoxin-1

Authors

  • Clifford Clark,

    1. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada,
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  • Darrin Bast,

    1. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada,
    2. Departments of Medicine and Microbiology, University of Toronto,
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  • Allan M. Sharp,

    1. Department of Medical Microbiology and Infectious Diseases, University of Alberta, 1–41 Medical Science Building, Edmonton, T6G 2H7, Canada,
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  • Phaedria M. St. Hilaire,

    1. Department of Chemistry, Duke University, Durham, North Carolina 27708-0346, USA,
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  • Rummana Agha,

    1. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada,
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  • Penelope E. Stein,

    1. Department of Medical Microbiology and Infectious Diseases, University of Alberta, 1–41 Medical Science Building, Edmonton, T6G 2H7, Canada,
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  • Eric J. Toone,

    1. Department of Chemistry, Duke University, Durham, North Carolina 27708-0346, USA,
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  • Randy J. Read,

    1. Department of Medical Microbiology and Infectious Diseases, University of Alberta, 1–41 Medical Science Building, Edmonton, T6G 2H7, Canada,
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  • James L. Brunton

    1. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada,
    2. Departments of Medicine and Microbiology, University of Toronto,
    3. Department of Microbiology, The Toronto Hospital, NU13, 200 Elizabeth Street, Toronto, Ontario M5G 2C4, Canada
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James L. Brunton Tel. (416) 340 3183; Fax (416) 340 5047

Abstract

The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 105 compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galα1-4Galβ1-4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.

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