Role of G protein Coupling in Fe(ll)-Uptake in Bacteria

描述（由申请人提供）：铁对细胞功能至关重要，导致病原体及其宿主之间对其进行竞争。在肠道和胃中，病原体如螺杆菌、沙门氏菌和弯曲杆菌依赖于Fe（II）的吸收。虽然Fe（II）的摄取是至关重要的毒力，很少有人知道其吸收的机制。 该项目的目标是了解膜蛋白FeoB的功能，该蛋白是细菌吸收Fe（II）所必需的。FeoB既新颖又独特。值得注意的是，FeoB的氨基酸序列预测了一个GTP结合结构域，该结构域连接到一束几个推定的跨膜α-螺旋。基于这种设计，我们假设FeoB可能是G蛋白偶联受体和/或通道的原始祖先。我们将采用生化，遗传和生物物理工具来测试这一假设，并建立FeoB的Fe（II）吸收的作用。我们的工作结果对于了解病原体中的铁稳态非常重要，并可能使我们能够确定治疗微生物感染的新策略。 第一个目的是确定FeoB在Fe（II）吸收中的功能。我们表明，N-末端结构域的FeoB作为一个监管GTP α结合蛋白。然而，膜嵌入结构域的功能仍然未知。我们将结合联合收割机在体内Fe（II）的吸收实验，在体外测量FeoB的Fe（II）结合和Fe（II）的运输性能，以确定是否FeoB作为转运/通道或作为受体蛋白的功能。 第二个目的是确定FeoB的G蛋白在Fe（II）吸收中的功能，以及G蛋白的活性如何调节。我们将确定是否FeoB本身，而不是下游的目标是由N-末端结构域和Fe（II）是否可以修改G蛋白的活性。 第三个目的是确定我们在FeoB的N-末端结构域中发现的鸟嘌呤核苷酸交换因子样活性的分子基础。我们将通过诱变禁用此活性，并确定核苷酸交换对体内Fe（II）摄取的重要性。 第四个目的是产生嵌入脂质双层中的FeoB晶体。最终，这将允许FeoB的可视化，并揭示其结构是否与其他G蛋白偶联的膜蛋白相关。

DESCRIPTION (provided by applicant): Iron is essential for cell function causing competition for it between pathogens and their hosts. In the gut and stomach, pathogens like Helicobacter, Salmonella and Campylobacter rely on the uptake of Fe(II). Although Fe(II)-uptake is critical for virulence, little is known about the mechanisms of its uptake. The goal of this project is to understand the function of the membrane protein FeoB which is essential for Fe(II)-uptake in bacteria. FeoB is both novel and unique. Notably, the amino acid sequence of FeoB predicts a GTP-binding domain that is connected to a bundle of several putative transmembrane alpha-helices. Based on this design, we hypothesize that FeoB may have served as primordial ancestor for G protein-coupled receptors and/or channels. We will employ biochemical, genetic and biophysical tools to test this hypothesis and to establish the role of FeoB for Fe(II) uptake. The results of our work are important for understanding iron homeostasis in pathogens and may enable us to identify new strategies for treating microbial infections. The first aim is to determine the function of FeoB in Fe(II) uptake. We show that the N-terminal domain of FeoB acts as a regulatory GTP alpha-binding protein. However, the function of the membrane embedded domain remains unknown. We will combine in vivo Fe(II) uptake experiments with in vitro measurements of FeoB's Fe(II)-binding and Fe(II)-transport properties to establish whether FeoB functions as transporter/channel or acts as a receptor protein. The second aim is to determine the function of FeoB's G protein in Fe(II)-uptake, and how the activity of the G protein is regulated. We will determine whether FeoB itself rather than a downstream target is regulated by the N-terminal domain and whether Fe(II) can modify the activity of the G protein. The third aim is to identify the molecular basis for a guanine-nucleotide-exchange-factor like activity that we discovered in FeoB's N-terminal domain. We will disable this activity by mutagenesis and determine the importance of nucleotide exchange for Fe(II) uptake in vivo. The fourth aim is to generate crystals of FeoB embedded in a lipid bilayer. Ultimately, this will allow visualization of FeoB, and reveal whether its structure is related to other G protein-coupled membrane proteins.