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Molecular Basis of PII function

PII 功能的分子基础

基本信息

批准号：
8070023
负责人：
GARY Paul ROBERTS
金额：
$ 30.97万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-01 至 2012-06-30
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：GlnB 及其同源物是 PII 蛋白家族的成员，并且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，并通过呈现两种形式的混合来做出反应：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许多受体蛋白的直接蛋白质-蛋白质相互作用来影响各种细胞过程。最后，许多氮过量形式的结构已得到解决。尽管了解了这些知识，一些非常核心的问题仍未解决，也是本提案的重点。首先，很明显，GlnB 样蛋白与 ATP 结合，但这种 ATP 结合尚未被认为具有生理重要性。然而，有力的初步证据表明，ATP 水平的变化会影响细胞中的 GlnB 功能，这意味着 GlnB 整合碳、氮和能量信号。研究能量状态对 GlnB 功能的作用是本提案的首要目标。目标 II 的重点是更好地了解目前未知的 GlnB 氮限制形式的结构。初步证据表明，它与已知的氮过量形式的结构有很大不同。 NMR 和 X 射线晶体学的直接结构分析将得到构象改变的 GlnB 变体的存在的补充。这一目标还直接测试了 1-酮戊二酸结合位点的最新模型，这对于通过遗传和生化方法的结合实现形式信号氮限制是必要的。最后，Aim III 解决了 GlnB 与 AmtB 的相互作用，AmtB 是一种完整的膜蛋白，也充当氨气通道。这种相互作用的一个重要结果是直接影响细胞中的 GlnB 水平，从而影响 GlnB 与其他蛋白质相互作用的能力。生化生理测定的组合将确定这种 GlnB-AmtB 相互作用调节的性质和重要性。所有这些目标成功的可能性非常高，因为必要的蛋白质变体已经在手，并且生化和遗传工具也可用。拟议实验的结果将极大地改进对所有生物体中 GlnB 同源物如何在分子水平上整合氮、碳和能量状态信号的描述。该项目解决了几乎所有原核生物中发现的 PII 蛋白系统对碳、氮和能量传感的交叉问题。由于其中心地位，它直接或间接影响有益（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病原生物如何感知和响应其环境。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum.

DOI：
10.1073/pnas.0306763101
发表时间：
2004-03
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Yaoping Zhang;E. Pohlmann;G. Roberts
通讯作者：
Yaoping Zhang;E. Pohlmann;G. Roberts

Identification and functional characterization of NifA variants that are independent of GlnB activation in the photosynthetic bacterium Rhodospirillum rubrum.