Structural and Functional Analysis of the Signal-Transducing Linker in the pH-Responsive One-Component System CadC of Escherichia coli
Structural and Functional Analysis of the Signal-Transducing Linker in the pH-Responsive One-Component System CadC of Escherichia coli
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DOI：
10.1016/j.jmb.2015.05.001
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发表时间：
2015-07-31
影响因子：
5.6
通讯作者：
Jung, Kirsten
中科院分区：
文献类型：
--
作者：
Buchner, Sophie;Schlundt, Andreas;Jung, Kirsten
在大肠杆菌中，pH响应的单组分信号系统CADC属于类ToxR蛋白家族，其成员具有保守的模块化结构，其N端的胞质翼状螺旋-转角螺旋DNA结合域紧随其后的是单个跨膜螺旋和C端的周质pH敏感域。在大肠杆菌CADC中，一个由大约50个氨基酸组成的细胞质连接子是将信号从传感器传递到DNA结合域所必需的。然而，对其转导机制却知之甚少。利用核磁共振波谱，我们在这里证明了连接区在溶液中本质上是无序的。此外，突变分析表明，它耐受一系列氨基酸取代(改变极性、刚性和a-螺旋形成倾向)，对延伸很健壮，但对截断敏感。事实上，截断要么逆转了目标操纵子cadBA的表达谱，要么完全将表达与外部pH解偶联。CADC通过其周质结构域进行二聚，但光散射分析没有提供分离的DNA结合结构域二聚的证据，无论有没有连接区。然而，细菌双杂交分析表明，CADC以一种依赖于刺激和连接子的方式形成稳定的二聚体，仅在pH&6.8时相互作用。值得注意的是，缺乏大部分连接子的反转cdBA表达谱的变体，在较高的pH时优先二聚化。因此，我们认为无序的CADC连接子是将周质传感器的pH依赖反应转化为结构重排所必需的，这有助于细胞质CADC DNA结合域的二聚化。(C)2015爱思唯尔有限公司。保留所有权利。
The pH-responsive one-component signaling system CadC in Escherichia coli belongs to the family of ToxR-like proteins, whose members share a conserved modular structure, with an N-terminal cytoplasmic winged helix-turn-helix DNA-binding domain being followed by a single transmembrane helix and a C-terminal periplasmic pH-sensing domain. In E. coli CadC, a cytoplasmic linker comprising approximately 50 amino acids is essential for transmission of the signal from the sensor to the DNA-binding domain. However, the mechanism of transduction is poorly understood. Using NMR spectroscopy, we demonstrate here that the linker region is intrinsically disordered in solution. Furthermore, mutational analyses showed that it tolerates a range of amino acid substitutions (altering polarity, rigidity and a-helix-forming propensity), is robust to extension but is sensitive to truncation. Indeed, truncations either reversed the expression profile of the target operon cadBA or decoupled expression from external pH altogether. CadC dimerizes via its periplasmic domain, but light-scattering analysis provided no evidence for dimerization of the isolated DNA-binding domain, with or without the linker region. However, bacterial two-hybrid analysis revealed that CadC forms stable dimers in a stimulus- and linker-dependent manner, interacting only at pH < 6.8. Strikingly, a variant with inversed cadBA expression profile, which lacks most of the linker, dimerizes preferentially at higher pH. Thus, we propose that the disordered CadC linker is required for transducing the pH-dependent response of the periplasmic sensor into a structural rearrangement that facilitates dimerization of the cytoplasmic CadC DNA-binding domain. (C) 2015 Elsevier Ltd. All rights reserved.