St Hilaire P M, Boyd M K, Toone E J
Department of Chemistry, Duke University, Durham, North Carolina 27708-0346.
Biochemistry. 1994 Dec 6;33(48):14452-63. doi: 10.1021/bi00252a011.
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.
开展了一项关于志贺样毒素1（SLT-1）与P(k)三糖[4-O-(4-O-α-D-吡喃半乳糖基)-4-O-β-D-吡喃葡萄糖苷甲酯]及其组成二糖结合的研究。该三糖代表SLT-1的中性糖脂受体球三糖神经酰胺（GbOse3）的碳水化合物识别结构域。可溶性三糖与可溶性五聚体B亚基的结合常数较弱，B亚基单体的K(a)为(0.5 - 1)×10³ M⁻¹。对结合数据的Scatchard分析表明，每个B亚基五聚体有五个相同的非相互作用碳水化合物结合位点，且结合过程中无协同性。尽管结合较弱（ΔG = -3.6 kcal mol⁻¹），但结合焓（ΔH = -12 kcal mol⁻¹）以及结合时摩尔热容的变化（ΔC(p) = -40 eu）与其他蛋白质-碳水化合物相互作用相当。动态光散射研究表明，碳水化合物结合会诱导蛋白质聚集。在超过90%的B亚基单体被结合的碳水化合物浓度下，远紫外圆二色光谱未发生变化，而近紫外圆二色光谱在270 nm附近出现变化，通过滴定得到的表观结合常数与等温滴定量热法获得的结果吻合良好。稳态荧光和荧光寿命测量表明，在糖类结合过程中，中心色氨酸的环境受到扰动，且这些变化与蛋白质聚集程度相关。基于结合热力学、光谱学以及结合诱导的聚集现象，我们提出了一种SLT-1与膜相互作用的模型，该模型依赖于蛋白质-碳水化合物相互作用实现特异性，依赖于蛋白质-脂质相互作用实现紧密结合。
