Phosphorylation of the response regulator CheV is required for adaptation to attractants during Bacillus subtilis chemotaxis
Phosphorylation of the response regulator CheV is required for adaptation to attractants during Bacillus subtilis chemotaxis
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DOI：
10.1074/jbc.m104955200
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发表时间：
2001-11-23
影响因子：
4.8
通讯作者：
Ordal, GW
中科院分区：
文献类型：
--
作者：
Karatan, E;Saulmon, MM;Ordal, GW
在革兰氏阳性土壤细菌枯草芽孢杆菌中，化学感受器通过两种蛋白质 CheW 和 CheV 与中心双组分激酶 CheA 偶联。 CheV 是一种双结构域蛋白，具有 N 端 CheW 样结构域和 C 端双组分受体结构域。在这项研究中，我们证明，在磷酸化 CheA (CheA-P) 存在的情况下，CheV 在体外被磷酸化在保守的天冬氨酸上。与 CheA-P 和系统的另一种响应调节剂 CheB 之间的磷酸转移反应相比，该反应较慢。与 CheB-P 相比，CheV-P 的稳定性也很高。与仅由受体结构域组成的截短形式相比，这两个特性在全长蛋白中更为明显，也就是说，CheW 样结构域的删除导致磷酸转移反应速率增加和磷酸化蛋白稳定性降低。 CheV 需要磷酸化才能适应化学引诱剂天冬酰胺的添加。在系留细胞测定中，表达 cheV 的不可磷酸化点突变体或缺乏整个受体结构域的截短突变体的菌株在适应添加天冬酰胺方面受到严重损害。然而，这两种菌株都显示出接近正常的逆时针偏向，表明在没有引诱剂的情况下，化学感受器通过突变的 CheV 蛋白有效地与 CheA 激酶偶联。 CheV 的 CheW 样结构域无法支持对添加天冬酰胺的完全适应，这也表明与 CheW 不同，该结构域本身可能导致信号复合物的形成，该复合物在引诱剂存在的情况下保持过度活跃。讨论了此功能的可能结构基础。
In the Gram-positive soil bacterium Bacillus subtilis, the chemoreceptors are coupled to the central two-component kinase CheA via two proteins, CheW and CheV. CheV is a two-domain protein with an N-terminal CheW-like domain and a C-terminal two-component receiver domain. In this study, we show that CheV is phosphorylated in vitro on a conserved aspartate in the presence of phosphorylated CheA (CheA-P). This reaction is slower compared with the phospho-transfer reaction between CheA-P and one other response regulator of the system, CheB. CheV-P is also highly stable in comparison with CheB-P. Both of these properties are more pronounced in the full-length protein compared with a truncated form composed only of the receiver domain, that is, deletion of the CheW-like domain results in increase in the rate of the phospho-transfer reaction and decrease in stability of the phosphorylated protein. Phosphorylation of CheV is required for adaptation to the addition of the chemoattractant asparagine. In tethered-cell assays, strains expressing an unphosphorylatable point mutant of cheV or a truncated mutant lacking the entire receiver domain are severely impaired in adaptation to the addition of asparagine. Both of these strains, however, show near normal counterclockwise biases, suggesting that in the absence of the attractant the chemoreceptors are efficiently coupled to CheA kinase by the mutant CheV proteins. Inability of the CheW-like domain of CheV to support complete adaptation to the addition of asparagine also suggests that unlike CheW this domain by itself may lead to the formation of signaling complexes that stay overactive in the presence of the attractant. A possible structural basis for this feature is discussed.