Phosphorylation triggers domain separation in the DNA binding response regulator NarL
Phosphorylation triggers domain separation in the DNA binding response regulator NarL
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DOI：
10.1021/bi0272205
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发表时间：
2003-03-11
期刊：
影响因子：
2.9
通讯作者：
Hubbell, WL
中科院分区：
文献类型：
--
作者：
Zhang, JH;Xiao, GP;Hubbell, WL
微生物双组分信号转导系统中的DNA结合蛋白通过未知的机制被磷酸化激活。NarL是来自土栖性大肠杆菌的硝酸盐/亚硝酸盐信号转导系统的一个例子。NarL由N-和C-末端结构域组成，后者含有DNA结合元件。为了探索活化机制，在整个C-末端结构域的九个不同位点处引入单个氮氧侧链，一次一个，以监测三级结构和与N-末端结构域接触的表面的状态。此外，制备了三对双标记蛋白质，利用磁偶极相互作用来监测结构域间的距离。这些定点自旋标记研究的结果表明，在N-末端结构域的远端位点的磷酸化可能通过铰链弯曲运动触发结构域分离。这反过来又提出了在最近的晶体结构中描述的配置的C-末端结构域的关键元素对接的DNA靶。这些数据还意味着，一个单一的构象的非磷酸化NarL存在于溶液中，并没有可检测的平衡之间的封闭和开放的构象。
DNA binding proteins of two-component signal transduction systems in microorganisms are activated by phosphorylation through an unknown mechanism. NarL is an example from the nitrate/nitrite signal transduction system of Eschericia coli. NarL consists of N- and C-terminal domains, the latter of which contains the DNA binding elements. To explore the mechanism of activation, single nitroxide side chains were introduced, one at a time, at nine different sites throughout the C-terminal domain to monitor the tertiary structure and the status of the surface in contact with the N-terminal domain. In addition, three pairs of doubly labeled proteins were prepared to monitor the interdomain distance using the magnetic dipolar interaction. The results of these site-directed spin-labeling studies reveal that phosphorylation at a distant site in the N-terminal domain triggers domain separation, likely by a hinge-bending motion. This in turn presents key elements of the C-terminal domain for docking to the DNA target in the configuration described in the recent crystal structure. The data also imply that a single conformation of unphosphorylated NarL exists in solution, and there is no detectable equilibrium between the closed and open conformations.