Thermal Domain Motions of CheA Kinase in Solution: Disulfide Trapping Reveals the Motional Constraints Leading to Trans-autophosphorylation
Thermal Domain Motions of CheA Kinase in Solution: Disulfide Trapping Reveals the Motional Constraints Leading to Trans-autophosphorylation
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DOI：
10.1021/bi900033r
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发表时间：
2009-04-28
期刊：
影响因子：
2.9
通讯作者：
Falke, Joseph J.
中科院分区：
文献类型：
--
作者：
Gloor, Susan L.;Falke, Joseph J.
组氨酸激酶CheA是细菌趋化性信号簇的核心组成部分，其中跨膜受体调节CheA自身激酶活性。CheA是一个同源二聚体，两个相同的亚基中的每一个具有五个不同的结构域，具有不同的结构和功能。与受体结合酶一样，游离酶催化一种转自激酶反应，其中一个亚基的催化结构域（P4）磷酸化另一个亚基的底物结构域（P1）。CheA结构域运动的分子分析对CheA反式自磷酸化的机制、CheA组装成信号簇以及CheA活性的受体调节具有重要意义。在这个初步的研究中，我们采用二硫键捕获分析对域之间的碰撞，从而绘制出域运动的范围和动力学。33个功能性单半胱氨酸CheA突变体的库，所有保留正常的自激酶活性，用于分析对称结构域对之间的二聚体内碰撞。CheA的同源二聚体结构确保每个突变体含有一对对称的、表面暴露的半胱氨酸残基。通过二硫键形成捕获的半胱氨酸-半胱氨酸碰撞表明PI是最移动的CheA结构域，但也检测到大幅度的P2、P4和P5结构域运动。使用17个功能性双半胱氨酸CheA突变体的文库进一步分析PI的迁移率，其中每个突变体亚基在P1上的固定探针位置处具有一个半胱氨酸。在一个实施方案中，第一个半胱氨酸在不同的结构域上，第二个半胱氨酸在不同的结构域上。所得的CheA同源二聚体含有四个半胱氨酸残基，因此二硫键捕获产生多个产品，通过分配方法进行鉴定。结果表明，PI底物结构域与姐妹亚基中P4'催化结构域上的残基快速碰撞，但未检测到亚基内碰撞。这一观察结果为CheA的反式自磷酸化提供了一个直接的、运动的解释，解释了为什么P1-P2区域的长接头不会在二聚体中缠结，并对CheA功能的其他方面具有重要意义。最后，提出了一个工作模型的运动约束，限制PI结构域的姐妹亚基的P4'催化结构域附近的空间区域。
The histidine kinase CheA is a central component of the bacterial chemotaxis signaling cluster, in which transmembrane receptors regulate CheA autokinase activity. CheA is a homodimer, and each of the two identical subunits possesses five different domains with distinct structures and functions. The free enzyme, like the receptor-bound enzyme, catalyzes a trans-autokinase reaction in which the catalytic domain (P4) of one subunit phosphorylates the substrate domain (P1) of the other subunit. Molecular analysis of CheA domain motions has important implications for the mechanism of CheA trans-autophosphorylation, for CheA assembly into the signaling cluster and for receptor regulation of CheA activity. In this initial study of the free CheA dimer, we employ disulfide trapping to analyze collisions between pairs of domains, thereby mapping out the ranges and kinetics of domain motions. A library of 33 functional single-cysteine CheA mutants, all retaining normal autokinase activity, is used to analyze intradimer collisions between symmetric domain pairs. The homodimeric structure of CheA ensures that each mutant contains a pair of symmetric, surface-exposed cysteine residues. Cysteine-cysteine collisions trapped by disulfide bond formation indicate that PI is the most mobile CheA domain, but large amplitude P2, P4, and P5 domain motions are also detected. The mobility of PI is further analyzed using a library of 17 functional dicysteine CheA mutants, wherein each mutant subunit possesses one cysteine at a fixed probe position on the P1. domain and a second cysteine on a different domain. The resulting CheA homodimers contain four cysteine residues; thus disulfide trapping yields multiple products that are identified by assignment methods. The findings reveal that the PI substrate domain collides rapidly with residues on the P4' catalytic domain in the sister subunit, but no intrasubunit collisions are detected. This observation provides a direct, motional explanation for CheA trans-autophosphorylation, explains why the long linkers of the P1-P2 region do not become tangled in the dimer, and has important implications for other aspects of CheA function. Finally, a working model is proposed for the motional constraints that limit the PI domain to the region of space near the P4' catalytic domain of the sister subunit.