F-19 NMR-STUDIES OF THE D-GALACTOSE CHEMOSENSORY RECEPTOR .1. SUGAR BINDING YIELDS A GLOBAL STRUCTURAL-CHANGE
F-19 NMR-STUDIES OF THE D-GALACTOSE CHEMOSENSORY RECEPTOR .1. SUGAR BINDING YIELDS A GLOBAL STRUCTURAL-CHANGE
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DOI：
10.1021/bi00231a021
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发表时间：
1991-04-30
期刊：
影响因子：
2.9
通讯作者：
FALKE, JJ
中科院分区：
文献类型：
--
作者：
LUCK, LA;FALKE, JJ
大肠杆菌D-半乳糖和D-葡萄糖受体是一种水溶性糖结合蛋白，是这些糖的不同化学感受和运输途径中的第一组分。 当糖结合并诱导构象变化时发生受体的激活，这反过来又能够与特定的膜蛋白对接。 只有含有结合D-葡萄糖的活化受体的结构是已知的。 为了研究糖诱导的结构变化，我们使用F-19 NMR探测了受体分子中广泛分布的12个位点。 5个位点是通过掺入5-氟色氨酸探测的色氨酸位置;通过定点诱变分配所得F-19 NMR共振。 其他7个位点是通过掺入3-氟苯丙氨酸探测的苯丙氨酸位置。 观察到糖与底物结合裂缝的结合引发了通过F-19 NMR频率偏移在12个标记位点中的10个处检测到的全局结构变化。 其中两个改变的位点位于与结合糖分子接触的货车德瓦尔斯底物结合裂缝中。 其他八个改变的网站，特别是两个Escherichans和六个苯丙氨酸平均分布在两个受体结构域之间，远离裂缝，因此糖结合后经历变构结构变化。 结果是一致的模型，其中多个二级结构元件，已知的基板裂缝和蛋白质表面之间的延伸，糖结合到裂缝后，在其平均位置发生变化。 这种结构偶联提供了一种机制，通过该机制，糖与底物裂隙的结合可以在受体表面上的一个或多个对接位点处引起结构变化。
The Escherichia coli D-galactose and D-glucose receptor is an aqueous sugar-binding protein and the first component in the distinct chemosensory and transport pathways for these sugars. Activation of the receptor occurs when the sugar binds and induces a conformational change, which in turn enables docking to specific membrane proteins. Only the structure of the activated receptor containing bound D-glucose is known. To investigate the sugar-induced structural change, we have used F-19 NMR to probe 12 sites widely distributed in the receptor molecule. Five sites are tryptophan positions probed by incorporation of 5-fluorotryptophan; the resulting F-19 NMR resonances were assigned by site-directed mutagenesis. The other seven sites are phenylalanine positions probed by incorporation of 3-fluorophenylalanine. Sugar binding to the substrate binding cleft was observed to trigger a global structural change detected via F-19 NMR frequency shifts at 10 of the 12 labeled sites. Two of the altered sites lie in the substrate binding cleft in van der Waals contact with the bound sugar molecule. The other eight altered sites, specifically two tryptophans and six phenylalanines distributed equally between the two receptor domains, are distant from the cleft and therefore experience allosteric structural changes upon sugar binding. The results are consistent with a model in which multiple secondary structural elements, known to extend between the substrate cleft and the protein surface, undergo shifts in their average positions upon sugar binding to the cleft. Such structural coupling provides a mechanism by which sugar binding to the substrate cleft can cause structural changes at one or more docking sites on the receptor surface.