Perry Susan, Shearer Neil, Little Richard, Dixon Ray
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UK, UK.
J Mol Biol. 2005 Mar 4;346(4):935-49. doi: 10.1016/j.jmb.2004.12.033. Epub 2005 Jan 26.
The NifL regulatory protein controls transcription of nitrogen fixation genes in Azotobacter vinelandii by modulating the activity of the transcriptional activator NifA through direct protein-protein interactions. The ability of NifL to integrate the antagonistic signals of redox and nitrogen status is achieved via the involvement of discrete domains in signalling specific environmental cues. NifL senses the redox status via an FAD co-factor located within the amino-terminal PAS domain and responds to the fixed nitrogen status by interaction with the signal transduction protein GlnK, which binds to the C-terminal GHKL domain of NifL. The GHKL domain binds adenosine nucleotides and is similar to the core catalytic domain of the histidine protein kinases. Binding of ADP to this domain increases the inhibitory activity of NifL and the formation of protein complexes with NifA. This inhibition is antagonised by the binding of 2-oxoglutarate, a key metabolic signal of the carbon status, to the amino-terminal GAF domain of NifA. In this study we have examined the properties of three mutations within conserved residues in the GHKL domain of NifL that impair signal transduction. All three mutations decrease the affinity of NifL for ADP significantly, but the mutant proteins exhibit discrete properties. The N419D mutation prevents inhibition of NifA activity by NifL both in vivo and in vitro. In contrast, the G455A and G480A mutations eliminate the redox response, but the mutant proteins retain some sensitivity to the fixed nitrogen status and the ability to interact with the GlnK signal transduction protein. Our data suggest that the absence of the redox switch in the G455A and G480A mutants is a consequence of their inability to override the allosteric effect of 2-oxoglutarate on NifA activity. Overall, these results demonstrate that the binding of adenosine nucleotides to the GHKL domain of NifL plays an important role in counteracting the response of NifA to 2-oxoglutarate, under conditions that are inappropriate for nitrogen fixation.
NifL调节蛋白通过直接的蛋白质-蛋白质相互作用调节转录激活因子NifA的活性，从而控制维涅兰德固氮菌中固氮基因的转录。NifL通过离散结构域参与特定环境信号的传递，实现整合氧化还原和氮状态拮抗信号的能力。NifL通过位于氨基末端PAS结构域内的FAD辅因子感知氧化还原状态，并通过与信号转导蛋白GlnK相互作用来响应固定氮状态，GlnK与NifL的C末端GHKL结构域结合。GHKL结构域结合腺苷核苷酸，与组氨酸蛋白激酶的核心催化结构域相似。ADP与该结构域的结合增加了NifL的抑制活性以及与NifA形成蛋白质复合物的能力。2-氧代戊二酸（碳状态的关键代谢信号）与NifA的氨基末端GAF结构域结合，可拮抗这种抑制作用。在本研究中，我们检测了NifL的GHKL结构域中影响信号转导的三个保守残基突变的特性。所有这三个突变均显著降低了NifL对ADP的亲和力，但突变蛋白表现出不同的特性。N419D突变在体内和体外均阻止了NifL对NifA活性的抑制。相反，G455A和G480A突变消除了氧化还原反应，但突变蛋白对固定氮状态仍保留一定敏感性以及与GlnK信号转导蛋白相互作用的能力。我们的数据表明，G455A和G480A突变体中氧化还原开关的缺失是由于它们无法克服2-氧代戊二酸对NifA活性的变构效应。总体而言，这些结果表明，在不适合固氮的条件下，腺苷核苷酸与NifL的GHKL结构域结合在抵消NifA对2-氧代戊二酸的反应中起重要作用。
