Molecular Basis of PII function

描述（由申请人提供）：GlnB及其同源物是PII蛋白家族的成员，是几乎所有原核细胞（包括所有主要病原体）的中心代谢调节因子。人们普遍认为GlnB样蛋白感知细胞中的碳氮平衡，并通过假设两种形式的混合物进行响应：一种形式表示氮过量，另一种形式表示氮限制。然后，这两种形式通过与细胞中的许多受体蛋白直接蛋白质-蛋白质相互作用来影响各种细胞过程。最后，一些结构的氮过剩的形式已经解决了。尽管了解了这些情况，但一些非常核心的问题仍未得到解决，这些问题也是本提案的重点。首先，很明显GlnB样蛋白结合ATP，但这种ATP结合并不被认为具有生理重要性。然而，强有力的初步证据表明，ATP水平的变化会影响细胞中GlnB的功能，这意味着GlnB整合了碳、氮和能量信号。检查能量状态对GlnB功能的作用是本提案的第一个目的。目的II的重点是更好地了解目前未知的GlnB的氮限制形式的结构。初步证据表明，它与氮过量形式的已知结构有很大的不同。通过NMR和X射线晶体学的直接结构分析将通过构象改变的GlnB变体的存在来补充。该目的还直接测试了1-酮戊二酸结合位点的最新模型，该模型是通过遗传和生物化学方法的组合实现形式信号氮限制所必需的。最后，目标III解决了GlnB与AmtB的相互作用，AmtB是一种整合的膜蛋白，也可作为氨气通道。这种相互作用的一个重要结果是直接影响细胞中GlnB的水平，从而影响GlnB与其他蛋白质相互作用的能力。生物化学生理学测定的组合将确定该GlnB-AmtB相互作用的调节的性质和重要性。所有这些目标都有很高的成功可能性，因为必要的蛋白质变体已经在手，生物化学和遗传学工具已经可用。拟议的实验的结果将是一个显着改善的描述如何在所有生物体中的GlnB同系物整合信号的氮，碳和能量状态在分子水平上。该项目解决了几乎所有原核生物中发现的PII蛋白系统的碳，氮和能量传感的交叉点。由于其中心地位，它直接或间接地影响有益（抗生素生产）和有害（毒素生产）的微生物过程。因此，这些结果将有助于解释有益生物和致病生物如何感知和应对环境。

DESCRIPTION (provided by applicant): GlnB and its homologs are members of the PII protein family and are central metabolic regulators in almost all prokaryotic cells, including all major pathogens. It is generally agreed that GlnB-like proteins sense the carbon-nitrogen balance in the cell and respond by assuming a mixture of two forms: one form signals nitrogen excess and the other signals nitrogen limitation. These two forms then affect a variety of cellular process by direct protein-protein interactions with a number of receptor proteins in the cell. Finally, a number of structures of the nitrogen-excess form have been solved. Despite this knowledge, some very central issues remain unresolved and are the focus of this proposal. First, it is clear that GlnB-like proteins bind ATP, but this ATP-binding has not been thought to be of physiological importance. However, strong preliminary evidence suggests that changes in ATP levels affect GlnB function in the cell, which implies that GlnB integrates carbon, nitrogen and energy signals. Examining the role of energy status on GlnB function is the first aim of this proposal. The focus of Aim II is to better understand the currently unknown structure of the nitrogen-limitation form of GlnB. Preliminary evidence suggests that it differs in dramatic ways from the known structures of the nitrogen-excess form. Direct structural analysis by NMR and X-ray crystallography will be complemented by the existence of conformationally altered GlnB variants. This aim also directly tests the recent model of the binding site of 1-ketoglutarate, which is necessary for achieving the form signaling nitrogen limitation, by a combination of genetic and biochemical approaches. Finally, Aim III addresses the interaction of GlnB with AmtB, an integral membrane protein that also serves as an ammonia gas channel. An important result of this interaction is to directly affect GlnB levels in the cell and therefore GlnB's ability to interact with other proteins. A combination of biochemical physiological assays will determine the nature and importance of the regulation of this GlnB-AmtB interaction. All these aims have a very high likelihood of success because the necessary protein variants are already in hand and the biochemical and genetic tools are available. The result of the proposed experiments will be a dramatically improved description of how GlnB homologs in all organisms integrate signals of nitrogen, carbon and energy status at the molecular level. This project addresses the intersection of carbon, nitrogen and energy sensing by the PII protein system found in almost all prokaryotes. Because of its centrality, it directly or indirectly affects both beneficial (antibiotic production) and harmful (toxin production) microbial processes. The results will therefore help explain how both beneficial and pathogenic organisms sense and respond to their environment.