Blat Y, Eisenbach M
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Biochemistry. 1994 Feb 1;33(4):902-6. doi: 10.1021/bi00170a008.
Bacterial chemotaxis is accomplished by regulating the direction of flagellar rotation. The primary target of the control appears to be CheY, a diffusible clockwise-signal molecule which interacts with the switch at the base of the flagellar motor and causes clockwise rotation. The regulatory mechanism appears to be phosphorylation/dephosphorylation of CheY. Here we demonstrate that CheZ, which accelerates the dephosphorylation of CheY, binds to CheY (immobilized on CNBr-activated Sepharose beads), that the binding to phosphorylated CheY is higher by over 2 orders of magnitude than the binding to nonphosphorylated CheY, and that the binding to both the phosphorylated and nonphosphorylated forms of CheY is significantly higher in the presence of Mg2+. We also show that the mutant proteins CheY13DK, CheY57DE, and CheY109KR bind CheZ to the same extent as wild-type CheY. The extent of the binding of these mutant proteins was not, however, increased in the presence of acetyl phosphate, the phosphorylating agent. The results indicate that neither a conformation which has a clockwise-causing activity in vivo nor phosphorylation is sufficient, alone, for maximal binding of CheZ to CheY and that Mg2+ is required for the binding of these proteins as well as for the phosphorylation and dephosphorylation of CheY.
细菌趋化作用是通过调节鞭毛旋转方向来实现的。控制的主要靶点似乎是CheY，一种可扩散的顺时针信号分子，它与鞭毛马达基部的开关相互作用并导致顺时针旋转。调节机制似乎是CheY的磷酸化/去磷酸化。在这里，我们证明，加速CheY去磷酸化的CheZ与CheY（固定在溴化氰活化的琼脂糖珠上）结合，与磷酸化CheY的结合比与非磷酸化CheY的结合高2个数量级以上，并且在Mg2+存在下，与磷酸化和非磷酸化形式的CheY的结合都显著更高。我们还表明，突变蛋白CheY13DK、CheY57DE和CheY109KR与CheZ的结合程度与野生型CheY相同。然而，在磷酸化剂乙酰磷酸存在下，这些突变蛋白的结合程度并未增加。结果表明，在体内具有顺时针诱导活性的构象或磷酸化单独都不足以使CheZ与CheY最大程度结合，并且Mg2+是这些蛋白质结合以及CheY磷酸化和去磷酸化所必需的。
