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，包括细胞内金属，如锌、铜和铁，从细胞的角度来看不是“微量元素”，但在大多数细胞中通常维持在更高的水平（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控制细胞金属离子调节的基本原则的重要性。我们描述这些新原理的方法涉及金属受体的机制和结构表征，这些受体以金属依赖的方式打开和关闭基因。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开关事件。初步研究揭示了MerR和Fur家族蛋白的金属响应成员与DNA靶标结合的第一个晶体结构，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。具体目的是解决这些金属调节蛋白的结构，功能和分子机制的关键，意想不到的问题。拟议的实验将采用x射线晶体学，生物物理方法和单粒子电子显微镜来了解金属与调节蛋白的结合如何诱导RNA聚合酶启动子复合物的构象变化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而直接影响聚合酶活性的。这些生物物理开关机制对细胞内金属生理的影响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理和机制。

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. PUBLIC HEALTH RELEVANCE: This proposal focuses on the fundamental ways in which living cells sense and control the amount of essential nutrient metals such as copper zinc and iron. For instance, when cells need more metal, some of these sensors work by turning on metal uptake machinery, and when cells need to get rid of excess metal, other sensors work to turn on machinery that ejects metals from the cell. This control is important because an imbalance in cellular metal can lead to diseases involving infectious agents, liver disorders, diabetes and brain function.