Two residues in the T-loop of GlnK determine NifL-dependent nitrogen control of nif gene expression
Two residues in the T-loop of GlnK determine NifL-dependent nitrogen control of nif gene expression
复制标题
DOI：
10.1074/jbc.m001935200
复制
发表时间：
2000-12-08
影响因子：
4.8
通讯作者：
Merrick, M
中科院分区：
文献类型：
--
作者：
Arcondéguy, T;Lawson, D;Merrick, M
大肠杆菌P-II蛋白旁系同源物GInB和GlnK的X射线晶体学分析表明，它们共享一个可重叠的结构核心，但在T环构象上可能不同，T环是蛋白质的一个区域（残基37-54），已被证明对与其他蛋白质的相互作用很重要。在肺炎克雷伯氏菌中，已显示GlnK在调节NifL介导的NifA活性抑制中具有明确定义的功能，以响应氮状态，并且当从染色体表达时，GlnB不取代GlnB，因为K的T环。肺炎杆菌和E.大肠杆菌GlnB和GlnK仅在三个残基43、52和54处不同，我们使用了先前构建的异源系统，其中肺炎杆菌nifLA。在E.大肠杆菌，研究GlnK残基43，52，和54的NifLA相互作用的调节的重要性。通过GlnB的定点诱变，我们已经表明，残基54是在NifA活性调节的背景下的T-环中的单个最重要的氨基酸。此外，在残基54和43中仅两个变化的组合允许GlnB充当GlnK并完全解除NifA活性的NifL抑制。
X-ray crystallographic analysis of the Escherichia coli P-II protein paralogues GInB and GlnK has shown that they share a superimposable structural core but can differ in conformation of the T-loop, a region of the protein (residues 37-54) that has been shown to be important for interaction with other proteins. In Klebsiella pneumoniae GlnK has been shown to have a clearly defined function in regulating NifL-mediated inhibition of NifA activity in response to the nitrogen status, and GlnB, when expressed from the chromosome, does not substitute for GlnB Because the T-loops of K. pneumoniae and E. coli GlnB and GlnK differ at just three residues, 43, 52, and 54, we have used a previously constructed heterologous system, in which a pneumoniae nifLA. is expressed in E. coli, to investigate the importance of GlnK residues 43, 52, and 54 for regulation of the NifLA interaction. By site-directed mutagenesis of glnB we have shown that residue 54 is the single most important amino acid in the T-loop in the context of the regulation of NifA activity. Furthermore, a combination of just two changes, in residues 54 and 43, allows GlnB to function as GlnK and completely relieve NifL inhibition of NifA activity.