Tryptophan residues flanking the second transmembrane helix (TM2) set the signaling state of the Tar chemoreceptor
Tryptophan residues flanking the second transmembrane helix (TM2) set the signaling state of the Tar chemoreceptor
复制标题
DOI：
10.1021/bi048969d
复制
发表时间：
2005-02-01
期刊：
影响因子：
2.9
通讯作者：
Manson, MD
中科院分区：
文献类型：
--
作者：
Draheim, RR;Bormans, AF;Manson, MD
大肠杆菌的化学感受器是同质二聚体膜蛋白，聚集在细胞两极附近的斑块中。它们将环境刺激转化为控制鞭毛旋转的细胞内信号。受体的功能域被细胞膜物理地分开。化学效应结合到细胞外（外质）结构域，细胞质结构域介导信号传导和适应。这两个结构域通过连接它们的第二个跨膜螺旋（TM2）进行通信。在高丰度的受体Tar和Tsr中，TM2两侧有色氨酸残基，它们应该优先定位于磷脂双分子层的极性层和疏水层之间的界面区。为了研究TM2旁边的Trp残基的功能意义，我们使用定点诱变技术产生了W192A和W209A的替换。W192A蛋白在体内和体外均保持充分的活性，但在体外实验中，它使天冬氨酸的K-i增加了3倍。W209A替代物在体外消除了受体介导的CheA刺激，并导致体内适应性甲基化水平的提高。这种表型在某些方面模仿结合天冬氨酸时所见的变化。由于W209A的取代可能导致TM2的c端进一步向细胞质中突出，这些结果强化了天冬氨酸结合引起类似位移的假设，将Trp从残基206移动到残基212的每个位置产生了各种各样的Tar信号状态，这与跨膜信号的活塞模型的预测基本一致。这些受体都没有完全锁定在一种信号模式中，尽管大多数显示出明显的信号偏差。我们的研究结果表明，TM2侧的Trp残基，特别是Trp-209，在设定Tar受体的基线活性和配体敏感性方面是重要的。我们还得出结论，tyr210残基在这种控制中至少起辅助作用。
The chemoreceptors of Escherichia coli are homodimeric membrane proteins that cluster in patches near the cell poles. They convert environmental stimuli into intracellular signals that control flagellar rotation. The functional domains of a receptor are physically separated by the cell membrane. Chemoeffectors bind to the extracellular (periplasmic) domain, and the cytoplasmic domain mediates signaling and adaptation. These two domains communicate through the second transmembrane helix (TM2) that connects them. In the high-abundance receptors Tar and Tsr, TM2 is flanked by tryptophan residues, which should localize preferentially to the interfacial zone between the polar and hydrophobic layers of the phospholipid bilayer. To investigate the functional significance of the Trp residues that flank TM2 of Tar, we used site-directed mutagenesis to generate the W192A and W209A substitutions. The W192A protein retains full activity in vivo and in vitro, but it increases the K-i for aspartate in the in vitro assay 3-fold. The W209A replacement eliminates receptor-mediated stimulation of CheA in vitro, and it leads to an increased level of adaptive methylation in vivo. This phenotype in some respects mimics the changes seen upon binding aspartate. Since the W209A substitution may cause the C-terminus of TM2 to protrude farther into the cytoplasm, these results reinforce the hypothesis that aspartate binding causes a similar displacement, Moving Trp to each position from residue 206 to residue 212 generated a wide variety of Tar signaling states that are generally consistent with the predictions of the piston model of transmembrane signaling. None of these receptors was completely locked in one signaling mode, although most showed pronounced signaling biases. Our findings suggest that the Trp residues flanking TM2, especially Trp-209, are important in setting the baseline activity and ligand sensitivity of the Tar receptor. We also conclude that the Tyr-210 residue plays at least an auxiliary role in this control.