Picking W D, McCann J A, Nutikka A, Lingwood C A
Department of Biology, Saint Louis University, Missouri 63103, USA.
Biochemistry. 1999 Jun 1;38(22):7177-84. doi: 10.1021/bi982335n.
Verotoxins (VTs) from Escherichia coli elicit human vascular disease as a consequence of specific binding to globotriaosylceramide (Gb3) receptors on endothelial cell surfaces. Molecular models based on the VT1 crystal structure 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 published structural data from cocrystals of the VT1 B-subunit (VT1B) and an analogue of the sugar moiety of Gb3. In this study, fluorescence spectroscopy was used to test model-based predictions of the location of Gb3 binding on the B-subunit pentamer of VT1. Resonance energy transfer was used to calculate the distance from a coumarin probe used to replace the acyl tail of Gb3 and the single tryptophan residue (Trp34) present within each VT1B monomer. The observed energy transfer efficiency (greater than 95%) suggests that these two moieties are approximately 13.3 A apart when a single distance is assumed. This distance is consistent with proposed models for the fit of Gb3 within the "cleft site" of the VT1 B-subunit. When the distances from Trp34 to the other coumarinGb3 molecules (bound to each of the four remaining monomers within the VT1B pentamer) are taken into consideration, it appears likely that the coumarin-modified Gb3 analogue used in this study associates with the previously proposed receptor binding site II of VT1. This is consistent with an observed binding preference of VT2c for coumarinGb3. To provide additional information on the association of Gb3 with the VT1 B-subunit, the influence of Gb3 glycolipid binding on the accessibility of Trp34 to different quenching agents in solution was then examined. Taken together, the data suggest that coumarin-labeled Gb3 preferentially binds to site II on VT1 in a position that is consistent with the previously described molecular models.
大肠杆菌产生的志贺毒素（VTs）通过特异性结合内皮细胞表面的球三糖基神经酰胺（Gb3）受体引发人类血管疾病。先前基于VT1晶体结构的分子模型用于研究VT1和其他志贺毒素识别受体的结构基础。有趣的是，这些基于模型预测的糖脂与VT1的结合与最近发表的VT1 B亚基（VT1B）和Gb3糖部分类似物的共晶体结构数据有所不同。在本研究中，荧光光谱法用于测试基于模型预测的Gb3在VT1 B亚基五聚体上的结合位置。共振能量转移用于计算用于取代Gb3酰基尾部的香豆素探针与每个VT1B单体中存在的单个色氨酸残基（Trp34）之间的距离。观察到的能量转移效率（大于95%）表明，当假设单一距离时，这两个部分相距约13.3埃。这个距离与Gb3在VT1 B亚基“裂隙位点”内的拟合模型一致。当考虑从Trp34到其他香豆素 - Gb3分子（与VT1B五聚体中其余四个单体中的每一个结合）的距离时，本研究中使用的香豆素修饰的Gb3类似物似乎与先前提出的VT1受体结合位点II相关。这与观察到的VT2c对香豆素 - Gb3的结合偏好一致。为了提供关于Gb3与VT1 B亚基结合的更多信息，随后研究了Gb3糖脂结合对溶液中Trp34与不同淬灭剂可及性的影响。综合来看，数据表明香豆素标记的Gb3优先结合在VT1上的位点II，其位置与先前描述的分子模型一致。
