Garzón A, Parkinson J S
Biology Department, University of Utah, Salt Lake City 84112, USA.
J Bacteriol. 1996 Dec;178(23):6752-8. doi: 10.1128/jb.178.23.6752-6758.1996.
CheA is a histidine kinase central to the signal transduction pathway for chemotaxis in Escherichia coli. CheA autophosphorylates at His-48, with ATP as the phosphodonor, and then donates its phosphoryl groups to two aspartate autokinases, CheY and CheB. Phospho-CheY controls the flagellar motors, whereas phospho-CheB participates in sensory adaptation. Polypeptides encompassing the N-terminal P1 domain of CheA can be transphosphorylated in vitro by the CheA catalytic domain and yet have no deleterious effect on chemotactic ability when expressed at high levels in wild-type cells. To find out why, we examined the effects of a purified P1 fragment, CheA[1-149], on CheA-related signaling activities in vitro and devised in vivo assays for those same activities. Although readily phosphorylated by CheA[260-537], the CheA catalytic domain, CheA[1-149], was a poor substrate for transphosphorylation by full-length CheA molecules, implying that the resident P1 domain monopolizes the CheA catalytic center. CheA-H48Q, a nonphosphorylatable mutant, failed to transphosphorylate CheA[1-149], suggesting that phosphorylation of the P1 domain in cis may alleviate the exclusion effect. In agreement with these findings, a 40-fold excess of CheA[1-149] fragments did not impair the CheA autophosphorylation reaction. CheA[1-149] did acquire phosphoryl groups via reversible phosphotransfer reactions with CheB and CheY molecules. An H48Q mutant of CheA[1-149] could not participate in these reactions, indicating that His-48 is probably the substrate site. The low level of efficiency of these phosphotransfer reactions and the inability of CheA[1-149] to interfere with CheA autophosphorylation most likely account for the failure of liberated P1 domains to jam chemotactic signaling in wild-type cells. However, an excess of CheA[1-149] fragments was able to support chemotactic signaling by P1-deficient cheA mutants, demonstrating that CheA[1-149] fragments have both transphosphorylation and phosphotransfer capability in vivo.
CheA是大肠杆菌趋化作用信号转导途径中的一种组氨酸激酶。CheA在His-48位点自磷酸化，以ATP作为磷供体，然后将其磷酸基团转移给两种天冬氨酸自激酶CheY和CheB。磷酸化的CheY控制鞭毛马达，而磷酸化的CheB参与感觉适应。包含CheA N端P1结构域的多肽在体外可被CheA催化结构域转磷酸化，但在野生型细胞中高水平表达时对趋化能力没有有害影响。为了弄清楚原因，我们检测了纯化的P1片段CheA[1-149]对体外CheA相关信号活性的影响，并设计了针对这些相同活性的体内检测方法。尽管CheA[1-149]很容易被CheA催化结构域CheA[260-537]磷酸化，但它却是全长CheA分子进行转磷酸化的不良底物，这意味着驻留的P1结构域垄断了CheA催化中心。CheA-H48Q是一种不可磷酸化的突变体，无法对CheA[1-149]进行转磷酸化，这表明顺式P1结构域的磷酸化可能会减轻这种排斥效应。与这些发现一致，过量40倍的CheA[1-149]片段不会损害CheA自磷酸化反应。CheA[1-149]确实通过与CheB和CheY分子的可逆磷酸转移反应获得了磷酸基团。CheA[1-149]的H48Q突变体无法参与这些反应，表明His-48可能是底物位点。这些磷酸转移反应的低效率以及CheA[1-149]无法干扰CheA自磷酸化，很可能解释了游离的P1结构域未能在野生型细胞中阻碍趋化信号传导的原因。然而，过量的CheA[1-149]片段能够支持缺乏P1结构域的cheA突变体的趋化信号传导，这表明CheA[1-149]片段在体内具有转磷酸化和磷酸转移能力。
