Molecular basis of PII function

GlnB及其同系物是几乎所有原核细胞中氮和碳状态信号的整合因子， 包括所有主要病原体。GlnB可以以两种极端形式存在，一种是氮充足的信号，另一种是 一种缺氮的信号。这些形式之间的平衡受到结合(富碳)的影响。 2-酮戊二酸和三磷酸腺苷，以及通过共价修饰GlnB来响应低水平的(富氮) 谷氨酰胺。这些不同形式的GlnB然后与一组关键的受体蛋白差异地相互作用，这些受体蛋白 控制氮代谢。GlnB内控制这一过程的信号转导机制是 了解很少，我们将通过生化和生理学获得关于这一过程的信息 对我们获得的一些引人注目的GlnB变体进行了分析。这些GlnB变体包括(I)变体 向表示缺氮的形式移动；(Ii)向表示缺氮的形式移动的变体 信号氮充足；以及(Iii)与ATP和2- 酮戊二酸。这些变异体将通过体外小分子结合、相互作用的测试进行检验。 受体和构象变化。这些变异将在体内进行检查，以确定准确的 体外测定对生理生化特性的影响。所有这些影响都将在 根据现有的和建议的GlnB构象和结构分析。这种方法有很高的 成功的可能性，因为有趣的变种已经在手中，生化和遗传 工具是可用的。我们还将研究GlnB监管的一个相关方面，即将其从 细胞质通过与膜相关的AmtB相互作用。我们已经开发了用于直接进入的工具 这种相互作用的体外分析和信息将与关于GlnB本身的上述数据整合在一起。 拟议中的实验结果将极大地改善对GlnB同源基因如何在 所有生物体都在分子水平上整合了氮、碳和能量状态的信号。这些信息 是非常重要的，因为这样的信号整合对于有益的(抗生素生产)和 有害的(毒素产生)微生物过程。因此，我们将更好地理解如何既有益于 病原生物体感知并对环境做出反应。

GlnB and its homologs are integrators of signals of nitrogen and carbon status in almost all prokaryotic cells, including all major pathogens. GlnB can exist in two extreme forms, one that signals nitrogen sufficiency and one that signals nitrogen deficiency. The equlibrium between those forms is affected by binding (carbon-rich) 2-ketoglutarate and ATP, and by covalent modification of GlnB in response to low levels of (nitrogen-rich) glutamine. These various forms of GlnB then interact differentially with a critical set of receptor proteins that control nitrogen metabolism. The signal transduction mechanism within GlnB that controls this process is poorly understood and we will gain information about this process through the biochemical and physiological analysis of some striking GlnB variants that we have obtained. These GlnB variants include (i) variants that are shifted toward the form that signals nitrogen deficiency; (ii) variants that are shifted toward the form that signals nitrogen sufficiency; and (iii) variants that are differentially altered in their affinities for ATP and 2- ketoglutarate. These variants will be examined by in vitro assays for small molecule binding, interaction with receptors and conformational changes. The variants will be examined in vivo for to determine the precise physiological impact of the biochemical properties measured in vitro. All of these effects will be interpreted in light of existing and proposed analysis of GlnB conformation and structure. The approach has a very high likelihood of success because the interesting variants are already in hand and the biochemical and genetic tools are available. We will also examine a related aspect of GlnB regulation, namely its removal from the cytoplasm by interaction with the membrane-associated AmtB. We have developed the tools for the direct in vitro analysis of this interaction and the information will be integrated with the above data on GlnB itself. 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. The information is broadly important because such signal integration is critical for both beneficial (antibiotic production) and harmful (toxin production) microbial processes. We will therefore understand better how both beneficial and pathogenic organisms sense and respond to their environment.