Localization of the binding site for modified Gb3 on verotoxin 1 using fluorescence analysis

Verotoxins (VTs) from Escherichia coli elicit human vascular disease as a c onsequence of specific binding to globotriaosylceramide (Gb(3)) receptors o n endothelial cell surfaces. Molecular models based on the VT1 crystal stru cture were used previously to investigate the structural basis for receptor recognition by VT1 and other verotoxins, Interestingly, these model-based predictions of glycolipid binding to VT1 differ somewhat from recently publ ished structural data from cocrystals of the VT1 B-subunit (VT1B) and an an alogue of the sugar moiety of Gb(3). in this study, fluorescence spectrosco py was used to test model-based predictions of the location of Gb(3) bindin g on the B-subunit pentamer of VT1. Resonance energy transfer was used to c alculate the distance from a coumarin probe used to replace the acyl tail o f Gb(3) and the single tryptophan residue (Trp34) present within each VT1B monomer. The observed energy transfer efficiency (greater than 95%) suggest s that these two moieties are approximately 13.3 Angstrom apart when a sing le distance is assumed. This distance is consistent with proposed models fo r the fit of Gb(3) within the "cleft site" of the VT1 B-subunit. When the d istances from Trp34 to the other coumarinGb(3) molecules (bound to each of the four remaining monomers within the VT1B pentamer) are taken into consid eration, it appears likely that the coumarin-modified Gb(3) analogue used i n this study associates with the previously proposed receptor binding site II of VT1. This is consistent with an observed binding preference of VT2c f or coumarinGb(3). To provide additional information on the association of G b(3) with the VT1 B-subunit, the influence of Gb(3) glycolipid binding on t he accessibility of Trp34 to different quenching agents in solution was the n examined. Taken together, the data suggest that coumarin-labeled Gb(3) pr eferentially binds to site II on VT1 in a position that is consistent with the previously described molecular models.