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

描述(申请人提供)：铁是细胞功能所必需的，导致病原体和它们的宿主之间对铁的竞争。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对Fe(II)的摄取。虽然Fe(II)吸收对毒力至关重要，但人们对其吸收机制知之甚少。 这个项目的目标是了解膜蛋白FeoB的功能，它是细菌吸收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，该结构域与几个假定的跨膜α-螺旋相连。基于这一设计，我们推测FeoB可能是G蛋白偶联受体和/或通道的原始祖先。我们将使用生化、遗传学和生物物理工具来验证这一假说，并确定FeoB在Fe(II)吸收中的作用。我们的工作结果对于理解病原体中的铁稳态是重要的，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)吸收中的作用。我们发现FeoB的N-末端结构域作为一个调节性的GTPα结合蛋白。然而，膜嵌入结构域的功能仍不清楚。我们将结合体内Fe(II)摄取实验和体外FeoB的Fe(II)结合和Fe(II)转运特性的测量来确定FeoB是作为转运体/通道还是作为受体蛋白发挥作用。 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蛋白的活性是如何调节的。我们将确定FeoB本身而不是下游靶点是否受N-末端结构域的调控，以及Fe(II)是否可以改变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。我们将通过突变来禁用这种活性，并确定核苷酸交换对体内Fe(II)摄取的重要性。 第四个目标是产生嵌入在脂质双层中的FeoB晶体。最终，这将使FeoB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.