Structural and thermodynamic characterization of the Escherichia coli RelBE toxin-antitoxin system: Indication for a functional role of differential stabilityt
Structural and thermodynamic characterization of the Escherichia coli RelBE toxin-antitoxin system: Indication for a functional role of differential stabilityt
复制标题
DOI：
10.1021/bi701037e
复制
发表时间：
2007-10-30
期刊：
影响因子：
2.9
通讯作者：
Gazit, Ehud
中科院分区：
文献类型：
--
作者：
Cherny, Izhack;Overgaard, Martin;Gazit, Ehud
RelE和RelB蛋白构成大肠杆菌relBE毒素-抗毒素系统的RNA干扰酶（毒素）及其同源抑制剂（抗毒素）组分。尽管该系统在大肠杆菌中的功能和生理活性得到了很好的描述。在大肠杆菌中，没有对蛋白质对在溶液中的折叠和稳定性进行结构研究。在这里，我们从结构和性能上表征了来自E.大肠杆菌在溶液中，无论是单独的，并在其复合状态。ROB抗毒素是一种根据圆二色性和红外光谱的α-螺旋蛋白，在溶液中形成低聚物，表现出高的热稳定性，T-M为58.5 ℃，具有相当大的耐热性，并且具有高的解折叠可逆性。相比之下，RelE毒素包括大部分的反平行P-折叠，显示出较低的热稳定性，T-M为52.5 ° C，并且表现出对热的异常敏感性。复合物的形成伴随着结构转变，导致T-M增加12摄氏度和显著的耐热性。此外，体内相互作用和蛋白质足迹实验表明，RelB的C-末端部分负责RelB-RelE相互作用，在其游离状态下是蛋白酶敏感的，而当与RelE复合时，其变得免受蛋白水解。总的来说，我们的研究结果支持这样的观点，即RelB在溶液中缺乏组织良好的疏水核心，而RelE是一个折叠良好的蛋白质。此外，我们的结果支持了E.在折叠和热力学性质上与ParD和CcdA抗毒素相似。的差异折叠状态的蛋白质进行了讨论，在其生理活动的背景下。
The RelE and RelB proteins constitute the RNA interferase (toxin) and its cognate inhibitor (antitoxin) components of the Escherichia coli relBE toxin-antitoxin system. Despite the well-described functionality and physiological activity of this system in E. coli, no structural study was performed on the folding and stability of the protein pair in solution. Here we structurally and thermodynamically characterize the ReIBE system components from E. coli in solution, both separately and in their complexed state. The ROB antitoxin, an alpha-helical protein according to circular dichroism and infrared spectroscopy, forms oligomers in solution, exhibits high thermostability with a T-M of 58.5 degrees C, has a considerable heat resistance, and has high unfolding reversibility. In contrasts the RelE toxin includes a large portion of antiparallel P-sheets, displays lower thermostability with a T-M of 52.5 degrees C, and exhibits exceptional sensitivity to heat. Complex formation, accompanied by a structural transition, leads to a 12 degrees C increase in the T-M and substantial heat resistance. Moreover, in vivo interaction and protein footprint experiments indicate that the C-terminal part of RelB is responsible for RelB-RelE interaction, being protease sensitive in its free state, while it becomes protected from proteolysis when complexed with RelE. Overall, our findings support the notion that RelB lacks a well-organized hydrophobic core in solution whereas RelE is a well-folded protein. Furthermore, our results support that RelB protein from E. coli is similar to ParD and CcdA antitoxins in both fold and thermodynamic properties. The differential folding state of the proteins is discussed in the context of their physiological activities.