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Localization of the binding site for modified Gb3 on verotoxin 1 using fluorescence analysis
W D Picking1, J A McCann, A Nutikka
1Department of Biology, Saint Louis University, Missouri 63103, USA. pickinwd@slu.edu
Biochemistry
|June 3, 1999
Summary
Verotoxins (VTs) bind to globotriaosylceramide (Gb3) receptors, causing vascular disease. This study uses fluorescence spectroscopy to confirm Gb3 binding sites on the VT1 B-subunit, supporting molecular models and revealing Gb3
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Verotoxins (VTs) from Escherichia coli induce human vascular disease through binding to globotriaosylceramide (Gb3) receptors on endothelial cells.
- Previous molecular models based on VT1 crystal structure predicted Gb3 binding sites, but recent cocrystal data present some discrepancies.
- Understanding the precise Gb3 binding location on the VT1 B-subunit is crucial for elucidating VT-mediated pathogenesis.
Purpose of the Study:
- To experimentally validate model-based predictions regarding the location of Gb3 binding on the VT1 B-subunit pentamer using fluorescence spectroscopy.
- To investigate the spatial relationship between Gb3 analogues and the tryptophan residue (Trp34) within the VT1 B-subunit monomers.
- To assess the influence of Gb3 binding on the accessibility of Trp34 to quenching agents, providing further insights into receptor-ligand interactions.
Main Methods:
- Utilized fluorescence spectroscopy, specifically resonance energy transfer (RET), to measure distances between a coumarin-labeled Gb3 analogue and the intrinsic Trp34 residue.
- Synthesized a coumarin probe replacing the acyl tail of Gb3 to facilitate fluorescence measurements.
- Examined the accessibility of Trp34 to various quenching agents in solution before and after Gb3 glycolipid binding.
Main Results:
- High energy transfer efficiency (>95%) indicated an approximate distance of 13.3 Å between the coumarin probe and Trp34, consistent with Gb3 binding in the proposed 'cleft site'.
- Analysis of distances to Gb3 molecules bound to other monomers suggested preferential association with receptor binding site II on VT1.
- Gb3 glycolipid binding altered the accessibility of Trp34 to quenching agents, further supporting specific interactions at the proposed binding site.
Conclusions:
- The experimental data strongly support the model-based predictions for Gb3 binding to site II on the VT1 B-subunit.
- Coumarin-labeled Gb3 preferentially binds to VT1 in a manner consistent with previously described molecular models, particularly within the 'cleft site'.
- These findings enhance the structural understanding of verotoxin-glycolipid interactions, relevant to the pathogenesis of VT-induced vascular disease.