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: 10083216
• 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/10083216
• 关键词: Address; Antibodies; 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; antiport; base; cofactor; computer studies; cooperative study; crosslink; dimer; genetic regulatory protein; human disease; in vitro Assay; mimetics; 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.

摘要 该项目阐述了阳离子中次级转运体的基本机制 扩散促进剂(CDF)家族携带过渡金属离子，如锌、锰、铁和钴 穿过薄膜。这些离子作为多种酶和酶的辅因子 调节蛋白。离子在许多不同的生理过程中发挥作用，作为一种 结果，CDF转运蛋白广泛存在。CDF转运体参与离子的摄取， 它们通常是环境和离子出口中的痕量元素，从而提供 对极端环境的容忍度。我们建议将结构性、功能性和 计算研究以产生对细菌锌离子的详细机理理解 并将这一理解推广到以 显示不同离子选择性的特定细菌和真核同源物 独特的结构域。目标1将专注于定义YIIP的构象变化 描述交替存取机制的特征，这是一种运送基质的范例 穿过生物膜。对于第一个目标，我们将使用冷冻-EM来表征 脂环境中YIIPP的外向态和无锌态的结构。 我们还将使用分子动力学来描述构象变化的动力学 这些状态之间以及运输周期的能量学之间的关系。AIM 2将调查 运输的功能决定因素。特别是，我们将研究使用In的YIIP的能量耦合。 体外转运试验，以表征锌离子转运与质子动力的偶联， 我将探索胞质结构域中锌离子结合部位的潜在作用 稳定同源二聚体的元件或作为调节活性的功能元件，并将 表征组成二聚体的两个分子之间的协作性。在《目标3》中，我们将 将我们对YIIP的研究扩展到来自不同生物的相关CDF家族成员， 因此对家谱的所有三个分支都进行了抽样。我们已经从之前的 一些细菌和真核生物的同源物已经被异源 在大肠杆菌或酿酒酵母中表达，用于基于细胞的分析。我们将对这些进行筛选 为同源基因的高表达水平和稳定性以及使用最佳表现进行探索奠定了基础 对于离子的选择性，比较能量耦合的机制，并评估功能 富含组氨酸的环的作用。这些环被认为是结合金属离子的，这表明 在调节或激活运输方面的潜在作用。