Metalloregulation by MerR and Fur Protein Families

描述（由申请人提供）：无机生理学研究中出现了许多新原理，包括从细胞角度来看，锌、铜和铁等细胞内金属不是“微量元素”，而是常规维持在大多数细胞中以高得多的水平（即，0.6 mM）。这些见解以及将金属生理学与许多疾病状态联系起来的新兴文献强调了建立管理细胞金属离子调节的基本原则的重要性。我们的方法来描绘这些新的原则涉及机械和金属受体的结构特征，开关和关闭基因在金属依赖的方式。该建议特别关注这些金属调节蛋白如何控制转录机制以实现特定类型的生理转换事件。初步研究揭示了MerR和Fur家族蛋白质与其DNA靶点结合的金属响应成员的第一个晶体结构，即CueR/DNA和Zur/DNA。新的结果提出了大量关于这些蛋白质如何控制细胞内金属离子稳态的问题。具体的目标是解决关键的，意想不到的问题，这些金属调节蛋白的结构，功能和分子机制。拟议的实验将采用X射线晶体学，生物物理学方法和单粒子电子显微镜，以了解如何金属结合到调节蛋白诱导构象变化的启动子复合物与RNA聚合酶，并导致基因表达的变化。这种方法将使我们能够理解金属结合事件是如何通过明确的蛋白质和核酸构象变化直接影响聚合酶活性来传达的。这些生物物理开关机制对细胞内金属生理学的影响，然后将使用新的单细胞分析方法进行检查，其总体目标是建立控制正常和疾病状态下金属离子稳态的一般原则和机制。

DESCRIPTION (provided by applicant): A number of new principles are emerging from the study of inorganic physiology, including the idea that intracellular metals such as zinc, copper and iron are not 'trace elements' from a cellular point of view, but are routinely maintained in most cells at much higher levels (i.e., 0.6 mM). These insights, as well as the emerging literature linking metal physiology to many disease states underscore the importance of establishing the fundamental principles governing cellular metal ion regulation. Our approach to delineating these new principles involves mechanistic and structural characterization of metal receptors that switch on and off genes in a metal dependent manner. This proposal specifically focuses on how such metalloregulatory proteins control the transcriptional machinery to achieve specific types of physiological switching events. Preliminary studies reveal the first crystal structures for metal-responsive members of the MerR and Fur family proteins bound to their DNA targets, namely CueR/DNA and Zur/DNA. The new results raise a significant number of questions about how these proteins control intracellular metal ion homeostasis. The specific aims are to resolve key, unanticipated questions about the structures, functions and molecular mechanisms of these metalloregulatory proteins. The proposed experiments will employ x-ray crystallography, biophysical methods and single particle electron microscopy to understand how metal binding to the regulatory protein induces conformational changes across the promoter complex with RNA polymerase and leads to changes in gene expression. This approach will enable us to understand how metal-binding events are communicated through explicit protein and nucleic acid conformation changes into a direct effect on polymerase activity. The effects of these biophysical switching mechanisms on intracellular metal physiology will then be examined using novel single cell analytical methods with the overarching goal of establishing general principles and mechanisms that control metal ion homeostasis in normal and disease states.