Interaction of the Shiga-like toxin type 1 B-subunit with its carbohydrate receptor.
A study of the binding of the Shiga-like toxin 1 (SLT-1) to the P(k) trisaccharide [methyl 4-O-(4-O-alpha-D-galactopyranosyl)-4-O-beta-D- glucopyranoside] and its constituent dissacharides was carried out. The trisaccharide represents the carbohydrate recognition domain of the neutral glycolipid receptor of the SLT-1, globotriosylceramide (GbOse3). The binding constant for soluble trisaccharide to the soluble pentameric B-subunit is weak, with a K(a) of (0.5-1) x 10(3) M-1 for B-subunit monomer. Scatchard analysis of the binding data indicates five identical non-interacting carbohydrate binding sites per B-subunit pentamer and no cooperativity in binding. Despite weak binding (delta G = -3.6 kcal mol-1), the enthalpy of binding (delta H = -12 kcal mol-1) and the change in molar heat capacity accompanying binding (delta C(p) = -40 eu) are comparable to other protein-carbohydrate interactions. Dynamic light scattering studies indicate that carbohydrate binding induces protein aggregation. At carbohydrate concentrations where > 90% of B-subunit monomers are bound, the far-UV CD spectra were unchanged, whereas a change in the near-UV CD, maximal near 270 nm, titrated to give an apparent binding constant in good agreement with that obtained by isothermal microcalorimetry. Steady-state fluorescence and fluorescence lifetime measurements indicated that the environments of the central tryptophans are perturbed during saccharide binding, and the changes correlate with the extent of protein aggregation. On the basis of the thermodynamics of binding, optical spectroscopy, and binding-induced aggregation, we propose a model of SLT-1-membrane interaction that relies on protein-carbohydrate interaction for specificity and protein-lipid interaction for tight binding.
St Hilaire
P M
PM
Department of Chemistry, Duke University, Durham, North Carolina 27708-0346.
Boyd
M K
MK
Toone
E J
EJ
D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D002240:Carbohydrate Sequence; D002942:Circular Dichroism; D004187:Disaccharides / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011940:Receptor Aggregation; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014281:Trihexosylceramides / Q000378:metabolism*; D014312:Trisaccharides / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D001427:Bacterial Toxins; D004187:Disaccharides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D014281:Trihexosylceramides; D014312:Trisaccharides; C018549:globotriaosylceramide; D014364:Tryptophan