Metal Ion Transport by the Cation Diffusion Facilitator Family

阳离子扩散促进剂家族的金属离子传输

• 批准号: 10319967
• 负责人: David L. Stokes
• 金额: $ 43.42万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319967
• 关键词: Address; Applications Grants; Binding; Binding Sites; Biological; Biological Assay; Biological Models; Cations; Cells; Coupled; Coupling; Cryoelectron Microscopy; Cytoplasmic Tail; Defect; Diffusion; Dimerization; Disease; Elements; Ensure; Environment; Enzymes; Escherichia coli; Family; Family member; Gel Chromatography; Genealogical Tree; Histidine; Homeostasis; Homologous Gene; Human Milk; Immunity; Immunoglobulin Fragments; In Vitro; Insulin; Ion Transport; Ions; Learning; Lipids; Measures; Membrane; Membrane Potentials; Membrane Proteins; Metal Ion Binding; Metals; Molecular; Molecular Conformation; Monitor; Mutagenesis; Neurotransmitters; Nutrient; Organism; Pathway interactions; Physiological Processes; Physiology; Play; Proteins; Protomer; Proton-Motive Force; Protons; Publications; Regulation; Regulatory Element; Role; Saccharomyces cerevisiae; Sampling; Secretory Vesicles; Seminal fluid; Series; Shewanella; Specificity; Structure; Synaptic Vesicles; Trace Elements; Transition Elements; Transport Process; Vesicle; Zinc; antibody mimetics; antiport; base; cofactor; computer studies; cooperative study; crosslink; dimer; genetic regulatory protein; human disease; in vitro Assay; molecular dynamics; novel therapeutics; pH gradient; sensor; stoichiometry; uptake

ABSTRACT This project addresses fundamental mechanisms by which secondary transporters in the Cation Diffusion Facilitator (CDF) family carry transition metal ions such as Zn2+, Mn2+, Fe2+ and Co2+ across the membrane. These ions serve as cofactors for a diverse array of enzymes and regulatory proteins. The ions play a role in many different physiological processes and, as a result, CDF transporters are widespread. CDF transporters are involved both in uptake of ions, which are normally trace elements in the environment, and in export of ions, thus providing tolerance to extreme environments. We propose to combine structural, functional and computational studies to generate a detailed mechanistic understanding of the bacterial Zn2+ transporter YiiP and to extend this understanding to other branches of the family represented by specific bacterial and eukaryotic homologs displaying different in ion selectivities and having unique structural domains. Aim 1 will focus on defining conformational changes in YiiP that characterize the alternating access mechanism, a paradigm for the transport of substrates across biological membranes. For this first aim, we will use cryo-EM to characterize the structure of the outward-facing state as well as Zn2+-free states of YiiP in a lipid environment. We will also use Molecular Dynamics to characterize the dynamics of conformational changes between these states as well as the energetics of the transport cycle. Aim 2 will investigate functional determinants of transport. In particular, we will study energy coupling of YiiP using in vitro transport assays to characterize the coupling of Zn2+ transport to the proton motive force, will explore potential roles of Zn2+ binding sites in the cytoplasmic domain as either structural elements stabilizing the homodimer or as functional elements that regulate activity, and will characterize cooperativity between the two molecules that compose the dimer. In Aim 3, we will expand our studies on YiiP to related CDF family members from a diverse array of organisms, thus sampling all three branches of the family tree. We have identified from previous publications a number of bacterial and eukaryotic homologs have been heterologously expressed in either E. coli or S. cerevisiae and used for cell-based assays. We will screen these homologs for high expression levels and stability and use the best behaved to explore the basis for ion selectivity, to compare mechanisms of energy coupling, and to evaluate the functional role of histidine-rich loops. These loops have been postulated to bind metal ions, suggesting potential roles in regulation or activation of transport.

抽象的 该项目研究了阳离子中二级转运蛋白的基本机制 扩散促进剂 (CDF) 系列携带过渡金属离子，例如 Zn2+、Mn2+、Fe2+ 和 Co2+ 穿过膜。这些离子作为多种酶的辅助因子， 调节蛋白。离子在许多不同的生理过程中发挥作用，并且作为 结果，CDF 转运蛋白广泛存在。 CDF 转运蛋白参与离子的摄取， 通常是环境中的微量元素，并以离子的形式输出，从而提供 对极端环境的耐受性。我们建议将结构、功能和 计算研究以深入了解细菌 Zn2+ 的机制 运输商 YiiP 并将这种理解扩展到以 特定的细菌和真核同系物表现出不同的离子选择性并具有 独特的结构域。目标 1 将重点定义 YiiP 中的构象变化， 描述交替存取机制的特征，这是基质传输的范例 穿过生物膜。为了实现第一个目标，我们将使用冷冻电镜来表征 脂质环境中 YiiP 的外向态和无 Zn2+ 态的结构。 我们还将使用分子动力学来表征构象变化的动力学 这些状态之间以及运输循环的能量学。目标 2 将进行调查 运输的功能决定因素。特别是，我们将研究 YiiP 的能量耦合，用于 体外转运测定来表征 Zn2+ 转运与质子动力的耦合， 将探索细胞质结构域中 Zn2+ 结合位点作为结构域的潜在作用 稳定同二聚体的元件或作为调节活性的功能元件，并且将 表征组成二聚体的两个分子之间的协同性。在目标 3 中，我们将 将我们对 YiiP 的研究扩展到来自多种生物体的相关 CDF 家族成员， 从而对家谱的所有三个分支进行采样。我们从之前的 出版物中许多细菌和真核同源物是异源的 在大肠杆菌或酿酒酵母中表达并用于基于细胞的测定。我们会筛选这些 同源物的高表达水平和稳定性并使用最佳表现来探索基础 用于离子选择性，比较能量耦合机制，并评估功能 富含组氨酸的环的作用。假设这些环可以结合金属离子，这表明 运输调节或激活中的潜在作用。