Rhodopsin activation exposes a key hydrophobic binding site for the transducin α-subunit C terminus
Rhodopsin activation exposes a key hydrophobic binding site for the transducin α-subunit C terminus
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DOI：
10.1074/jbc.m402567200
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发表时间：
2004-07-09
影响因子：
4.8
通讯作者：
Farrens, DL
中科院分区：
文献类型：
--
作者：
Janz, JM;Farrens, DL
构象变化使光感受器视紫红质与g蛋白转导蛋白偶联并激活。在这里，我们证明了这些蛋白质之间的关键相互作用发生在转导蛋白α亚基（G(Talpha)）的C端和受体激活期间暴露的视紫红质细胞质表面的疏水裂缝之间。我们通过用荧光探针双烷标记视紫红质突变体来绘制这种相互作用，然后评估G(Talpha) C末端（含有色氨酸猝灭基团）的肽类似物的结合如何影响其荧光。从这些和其他分析中，我们得出结论，G(Talpha) c端尾部以视网膜连接的方式与螺旋6的内面结合。此外，我们发现在暴露的间隙中包含关键残基的“疏水补丁”是转导蛋白结合/激活所必需的，因为它增强了对G(Talpha) c末端尾部的结合亲和力，对这种相互作用贡献高达3 kcal/mol。我们推测这里发现的疏水相互作用可能在其他GPCR信号系统中很重要，并且我们的色氨酸/双烷荧光方法可能通常用于绘制蛋白质-蛋白质相互作用的位点。
Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin alpha-subunit (G(Talpha)) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G(Talpha) C terminus ( containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G(Talpha) C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G(Talpha) C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/ bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.