McEvoy M M, Bren A, Eisenbach M, Dahlquist F W
Institute of Molecular Biology, University of Oregon, Eugene, OR, 97403, USA.
J Mol Biol. 1999 Jun 25;289(5):1423-33. doi: 10.1006/jmbi.1999.2830.
CheY is the response regulator protein serving as a phosphorylation-dependent switch in the bacterial chemotaxis signal transduction pathway. CheY has a number of proteins with which it interacts during the course of the signal transduction pathway. In the phosphorylated state, it interacts strongly with the phosphatase CheZ, and also the components of the flagellar motor switch complex, specifically with FliM. Previous work has characterized peptides consisting of small regions of CheZ and FliM which interact specifically with CheY. We have quantitatively measured the binding of these peptides to both unphosphorylated and phosphorylated CheY using fluorescence spectroscopy. There is a significant enhancement of the binding of these peptides to the phosphorylated form of CheY, suggesting that these peptides share much of the binding specificity of the intact targets of the phosphorylated form of CheY. We also have used modern nuclear magnetic resonance methods to characterize the sites of interaction of these peptides on CheY. We have found that the binding sites are overlapping and primarily consist of residues in the C-terminal portion of CheY. Both peptides affect the resonances of residues at the active site, indicating that the peptides may either bind directly at the active site or exert conformational influences that reach to the active site. The binding sites for the CheZ and FliM peptides also overlap with the previously characterized CheA binding interface. These results suggest that interaction with these three proteins of the signal transduction pathway are mutually exclusive. In addition, since these three proteins are sensitive to the phosphorylation state of CheY, it may be that the C-terminal region of CheY is most sensitive for the conformational changes occurring upon phosphorylation.
CheY是一种应答调节蛋白，在细菌趋化信号转导途径中作为磷酸化依赖性开关。在信号转导过程中，CheY与许多蛋白质相互作用。在磷酸化状态下，它与磷酸酶CheZ以及鞭毛马达开关复合体的组分强烈相互作用，特别是与FliM。先前的研究已对由CheZ和FliM的小区域组成的、与CheY特异性相互作用的肽进行了表征。我们使用荧光光谱法定量测量了这些肽与未磷酸化和磷酸化的CheY的结合。这些肽与磷酸化形式的CheY的结合有显著增强，这表明这些肽具有磷酸化形式的CheY完整靶标的许多结合特异性。我们还使用现代核磁共振方法来表征这些肽在CheY上的相互作用位点。我们发现结合位点是重叠的，主要由CheY C末端部分的残基组成。两种肽都会影响活性位点残基的共振，这表明这些肽可能直接结合在活性位点，或者产生延伸至活性位点的构象影响。CheZ和FliM肽的结合位点也与先前表征的CheA结合界面重叠。这些结果表明，与信号转导途径中这三种蛋白质的相互作用是相互排斥的。此外，由于这三种蛋白质对CheY的磷酸化状态敏感，可能CheY的C末端区域对磷酸化时发生的构象变化最为敏感。
