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.

摘要