Gloor Susan L, Falke Joseph J
Department of Chemistry and Biochemistry, University of Colorado, Boulder, 80309-0215, USA.
Biochemistry. 2009 Apr 28;48(16):3631-44. doi: 10.1021/bi900033r.
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 P1 is the most mobile CheA domain, but large amplitude P2, P4, and P5 domain motions are also detected. The mobility of P1 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 P1 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 P1 domain to the region of space near the P4' catalytic domain of the sister subunit.
组氨酸激酶CheA是细菌趋化信号簇的核心组成部分，其中跨膜受体调节CheA的自激酶活性。CheA是一种同型二聚体，两个相同的亚基各自拥有五个具有不同结构和功能的不同结构域。游离酶与受体结合酶一样，催化一种反式自激酶反应，其中一个亚基的催化结构域（P4）使另一个亚基的底物结构域（P1）磷酸化。对CheA结构域运动的分子分析对于CheA反式自磷酸化的机制、CheA组装到信号簇以及受体对CheA活性的调节具有重要意义。在对游离CheA二聚体的这项初步研究中，我们采用二硫键捕获来分析结构域对之间的碰撞，从而描绘出结构域运动的范围和动力学。一个包含33个功能性单半胱氨酸CheA突变体的文库，所有突变体都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的同型二聚体结构确保每个突变体都包含一对对称的、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P1是CheA中最具流动性的结构域，但也检测到了P2、P4和P5结构域的大幅度运动。使用一个包含17个功能性双半胱氨酸CheA突变体的文库进一步分析P1的流动性，其中每个突变体亚基在P1结构域的固定探针位置有一个半胱氨酸，在另一个不同结构域有第二个半胱氨酸。由此产生的CheA同型二聚体包含四个半胱氨酸残基；因此二硫键捕获产生多种产物，通过分配方法进行鉴定。研究结果表明，P1底物结构域与姐妹亚基中P4'催化结构域上的残基迅速碰撞，但未检测到亚基内碰撞。这一观察结果为CheA反式自磷酸化提供了直接的运动学解释，解释了为什么P1 - P2区域的长连接子在二聚体中不会缠结，并且对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，用于解释将P1结构域限制在姐妹亚基P4'催化结构域附近空间区域的运动限制。
