Modelling of the interaction of verotoxin-1 o(VT1) with its gly.colipid receptor, gl botriaosylceram=de (Gb3) Per-Georg Nyholm* Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada James L. Brunton Departments of Microbiology and Medicine, University of Toronto, The Toronto Hospital and the Samuel Lunenfeld Research Institute at Mount Sinai Hospital Toronto, Ontario M5S 1A 1, Canada and Clifford A. Lingwood Department of Microbiology, Hospital for Sick Children, Toronto, Ontario M5G 1)(8, and Departments of Clinical Biochemistry, Biochemistry and Microbiology, University of Toronto, Toronto, Ontario, Canada Received 20 September 1994; revised 2 January 1995 Possible binding sites for the glycolipid globotriaosylceramide (GalcO -*4Gal/~l -.4GIc/~1 -,1 Cer; Gb3) on the B-subunits of verotoxin-1 (VT1) were explored using binding data for specifically mutated verotoxins and by computational docking of favoured conformers of Gb3 with the crystal structure of VTI. Calculations using the GRID program suggested a site with favourable hydrophobic interactions at the exposed side chain of Phe30. One of the favoured conformers of Gb 3 was docked into this site, with the hydrophobic face of the internal Gal/~ residue in contact with the side chain of Phe30. After energy minimization, the two terminal saccharide residues of Gb3 (Gala and Gal/~) showed favourable interactions with the toxin. In the proposed model of the complex, the terminal Gala of Gb3 is located in proximity to aspartates 16-18 of VTI. The model is in agreement with available experimental binding data for the interaction of globoglycolipids with different naturally occurring and mutated verotoxins. Keywords: verotoxin; glycolipid; molecular recognition The verotoxins, also termed Shiga-like toxins, constitute a family of closely related toxins produced by certain strains of Escherichia coli bacteria 1. Several of these toxins are known to cause disease in humans and other mammals. Verotoxin-I (VT1) and possibly also verotoxin-2 (VT2) can give rise to serious kidney damage leading to the haemolytic uraemic syndrome (HUS) in infants 2. In pigs, another verotoxin, VT2e, has been described as giving rise to 'pig oedema disease '3. All the verotoxins consist of a pentamer of 7.5 kDa B-subunits and an A-subunit (32kDa) with catalytic activity 4. Binding of the toxin to the cell membrane of the target cell is mediated by the B-subunits. After internalization of the toxin, the A subunit switches off protein synthesis in the target cell by cleaving a specific adenine residue of ribosomal RNA 5. * To whom correspondence should be addressed Extensive in vitro binding studies have demonstrated that verotoxins interact with certain glycolipids of the globo- series 6"7. The formulae, abbreviations and specificities of these glycolipids are shown in Table 1. In particular, globotriaosylceramide (Gb3) is an efficient receptor for all verotoxins. Based on histochemical studies and experiments on cell lines, it has been concluded that Gb 3 located in the cell membranes of endothelial cells in the glomeruli constitutes the relevant receptor for verotoxins in the pathogenesis of HUS 8. The need for a rational treatment of HUS and the general interest in glycolipid/protein interactions has prompted detailed structural studies. The crystal structure of the B-subunits of VTI was determined by Stein et al. 9 and the crystal structure of the holotoxin was reported recently 1°. Furthermore, extensive work by site-directed mutagenesis has been performed to define the amino acid residues involved in the receptor interactions (Table 1) 11 13. However, despite these efforts, 0141-8130/95/$09.50!i: 1995 Elsevier ScienceB.V.All rights reserved Int. J. Biol. Macromol. Volume 17 Number 3-4 1995 199