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 该项目解决了阳离子中的二次转运蛋白的基本机制 扩散促进因（CDF）家族携带过渡金属离子，例如Zn2+，Mn2+，Fe2+和CO2+ 穿过整个膜。这些离子是各种酶的辅助因子， 调节蛋白。离子在许多不同的生理过程中发挥作用，作为一个 结果，CDF转运蛋白广泛。 CDF转运蛋白都参与了离子的摄取， 通常是环境中和离子出口中的跟踪元素，从而提供 对极端环境的容忍度。我们建议结合结构，功能和 计算研究以产生对细菌Zn2+的详细机械理解 运输者yiip并将这种理解扩展到由家族的其他分支 特定的细菌和真核同源物在离子选择性中显示不同，并且具有 独特的结构域。 AIM 1将专注于定义Yiip中的构象变化 表征交替的访问机制，这是基板运输的范例 整个生物膜。对于第一个目标，我们将使用Cryo-Em来表征 面向外向状态以及Zn2+的Zn2+YIIP状态在脂质环境中的结构。 我们还将使用分子动力学来表征构象变化的动力学 在这些状态以及运输周期的能量之间。 AIM 2将调查 运输的功能决定因素。特别是，我们将研究YIIP的能量耦合 体外传输分析以表征Zn2+传输与质子动力的耦合， 将探索Zn2+结合位点在细胞质结构域的潜在作用 稳定同型二聚体或作为调节活动的功能元素的要素，并将 表征组成二聚体的两个分子之间的合作性。在AIM 3中，我们将 将我们对YIIP的研究扩展到来自各种有机体的相关CDF家族成员， 因此，对家谱的所有三个分支进行了采样。我们已经从以前的 出版物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，用于基于细胞的测定。我们将筛选这些 高表达水平和稳定性的同源物，并利用最好的行为来探索基础 对于离子选择性，比较能量耦合机制并评估功能 富含组氨酸的回路的作用。这些循环已经假设结合金属离子，这表明 在调节或激活转运中的潜在作用。