Identification of an Anchor Residue for CheA-CheY Interactions in the Chemotaxis System of Escherichia coli
Identification of an Anchor Residue for CheA-CheY Interactions in the Chemotaxis System of Escherichia coli
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DOI：
10.1128/jb.00426-11
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发表时间：
2011-08-01
影响因子：
3.2
通讯作者：
Stewart, Richard C.
中科院分区：
文献类型：
--
作者：
Thakor, Hemang;Nicholas, Sarah;Stewart, Richard C.
自磷酸化CheA（P-CheA）的磷酰基转移到CheY是细菌趋化性信号转导途径中的重要步骤。该反应涉及CheY（i）与P-CheA的P2结构域结合，然后（ii）从P1结构域获得磷酰基。晶体结构表明在CheY-P2结合界面处存在许多侧链相互作用。为了研究参与这些接触的P2侧链的个体贡献，我们分析了八个丙氨酸取代突变对CheA-CheY结合相互作用的影响。P2中的F214 A取代导致CheA-CheY结合亲和力降低1,000倍，而P2其他位置的Ala取代对结合亲和力的影响很小（E171 A、E178 A和I216 A）或没有可检测的影响（H181 A、D202 A、D207 A和C213 A）。这些结果进行了讨论，与以前在硅片预测的热点和锚的位置在CheA-CheY接口。我们还研究了这些突变对活细胞中趋化性信号转导的影响。CheA（F214 A）在介导CheY-YFP定位于大肠杆菌细胞两极形成的大簇信号蛋白方面存在缺陷，而其他CheA变体在这方面与野生型（wt）CheA（CheA（wt））没有区别。在我们的一组突变体中，只有CheA（F214 A）在运动琼脂试验中表现出明显降低的支持趋化性的能力。然而，令人惊讶的是，在监测受体调节的磷酸化CheY产生的FRET测定中，CheA（F214 A）（和其他每种Ala取代突变体）的表现与CheA（wt）一样好。总体而言，我们的研究结果表明，F214作为一个锚残基在CheA-CheY界面，并作出了重要的贡献，在体外和体内的结合能，然而，这种贡献的损失并没有一个大的负面影响的信号通路的整体能力，以调节P-CheY水平，以响应于化学引诱剂。
Transfer of a phosphoryl group from autophosphorylated CheA (P-CheA) to CheY is an important step in the bacterial chemotaxis signal transduction pathway. This reaction involves CheY (i) binding to the P2 domain of P-CheA and then (ii) acquiring the phosphoryl group from the P1 domain. Crystal structures indicated numerous side chain interactions at the CheY-P2 binding interface. To investigate the individual contributions of the P2 side chains involved in these contacts, we analyzed the effects of eight alanine substitution mutations on CheA-CheY binding interactions. An F214A substitution in P2 caused similar to 1,000-fold reduction in CheA-CheY binding affinity, while Ala substitutions at other P2 positions had small effects (E171A, E178A, and I216A) or no detectable effects (H181A, D202A, D207A, and C213A) on binding affinity. These results are discussed in relation to previous in silico predictions of hot-spot and anchor positions at the CheA-CheY interface. We also investigated the consequences of these mutations for chemotaxis signal transduction in living cells. CheA(F214A) was defective in mediating localization of CheY-YFP to the large clusters of signaling proteins that form at the poles of Escherichia coli cells, while the other CheA variants did not differ from wild-type (wt) CheA (CheA(wt)) in this regard. In our set of mutants, only CheA(F214A) exhibited a markedly diminished ability to support chemotaxis in motility agar assays. Surprisingly, however, in FRET assays that monitored receptor-regulated production of phospho-CheY, CheA(F214A) (and each of the other Ala substitution mutants) performed just as well as CheA(wt). Overall, our findings indicate that F214 serves as an anchor residue at the CheA-CheY interface and makes an important contribution to the binding energy in vitro and in vivo; however, loss of this contribution does not have a large negative effect on the overall ability of the signaling pathway to modulate P-CheY levels in response to chemoattractants.