Identification of a novel two component system in Thermotoga maritima.: Complex stoichiometry and crystallization
Identification of a novel two component system in Thermotoga maritima.: Complex stoichiometry and crystallization
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DOI：
10.1016/j.bbapap.2007.02.005
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发表时间：
2007-05-01
影响因子：
3.2
通讯作者：
Marina, Alberto
中科院分区：
文献类型：
--
作者：
Casino, Patricia;Fernandez-Alvarez, Ana;Marina, Alberto
由组氨酸激酶及其同源响应调节器组成的双组分信号转导系统是微生物感知和响应许多不同环境条件变化的主要机制。不同的海栖热袍菌组氨酸激酶已被用作原型；其中，孤儿TM0853已被提出作为I类组氨酸激酶的结构模型。我们使用磷酸转移测定法将 TM0468 鉴定为 TM0853 的伙伴反应调节剂。由于全长 TM0853 可以在大肠杆菌中作为可溶性蛋白产生，因此首次用于分析完整的双组分系统中的联合化学计量。我们证明 TM0853 或其细胞质催化部分通过天然 PAGE 与 TM0468 形成 1:1 复合物。即使每个单独的蛋白质存在过量，复合带也是独特的，表明该联合是合作的。我们通过使用超速离心测定证实了这些发现。因此，我们提出，正统双组分系统中相互作用的一般模式可能是二聚组氨酸激酶和两个反应调节剂之间的化学计量和协作复合物。最后，我们已经能够生产 TM0853 和 TM0468 细胞质部分之间的复合物的蛋白质晶体，其衍射间距为 2.8 埃布拉格间距。 (C) 2007 Elsevier B.V. 保留所有权利。
Two-component signal transduction systems, comprised of histidine kinase and its cognate response regulator, are the predominant mechanism by which microorganisms sense and respond to changes in many different environmental conditions. Different Thermotoga maritima histidine kinases have been used as prototypes; among them, the orphan TM0853 has been presented as a structural model of class I histidine kinases. We used phosphotransfer assays to identify TM0468 as the partner response regulator of TM0853. Since full-length TM0853 can be produced as a soluble protein in Escherichia coli, it was used to analyze the union stoichiometry in an intact two-component system for the first time. We demonstrate that TM0853, or its cytoplasmic catalytic portion, form a 1: 1 complex with TM0468 with native PAGE. The complex band is unique, even in the presence of an excess of each individual protein, indicating that the union is cooperative. We corroborated these findings by using ultracentrifugation assays. Therefore, we propose that the general mode of interaction in an orthodox two-component system may be the stoichiometric and cooperative complex between a dimeric histidine kinase and two response regulators. Finally, we have been able to produce protein crystals of the complex between the cytoplasmic portion of TM0853 and TM0468 that diffract to 2.8 angstrom Bragg spacing. (C) 2007 Elsevier B.V. All rights reserved.