Mechanism of Divalent Metal Transport by NRamp-Family Transporters

NRamp 家族转运蛋白转运二价金属的机制

• 批准号: 9899469
• 负责人: RACHELLE GAUDET
• 金额: $ 4.46万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9899469
• 关键词: Administrative Supplement; Anemia; Autoimmune Diseases; Bacteria; Basic Science; Biochemical; Biological Assay; Blood; Brain; Cells; Coupled; Crystallization; Cytosol; Data; Diet; Disease; Energy Metabolism; Engineering; Event; Family; Funding; Goals; Health; Homeostasis; Homologous Gene; Human; Immunity; Ion Cotransport; Ion Transport; Ions; Iron; Knowledge; Liver; Manganese; Membrane Transport Proteins; Metabolic; Metals; Modeling; Molecular; Molecular Conformation; Natural Immunity; Neuraxis; Neurons; Nramp protein; Parkinson Disease; Pathogenicity; Pathologic; Physiological; Physiological Processes; Play; Protein Family; Protons; Research; Research Training; Resolution; Role; Sodium; Structure; Students; System; Therapeutic; Tissues; Transition Elements; X-Ray Crystallography; antiporter; deprotonation; divalent metal; drug development; in vitro activity; in vivo; insight; man; oxygen transport; pH gradient; pathogen; protonation; scaffold; simulation; symporter; uptake

Research Summary Metals such as iron and manganese are essential to many physiological processes including oxygen transport and energy metabolism. But overabundance of these metals is toxic, and their physiological levels are therefore tightly regulated. Nramps (natural resistance-associated macrophage proteins) are membrane transporters that import divalent metal ions into cells. Nramps are important for both divalent metal uptake from the diet and cellular import of metals into the cytosol. Nramps are therefore critical factors in maintaining homeostasis of divalent metals, particularly iron and manganese. Nramp proteins are conserved from bacteria to man, and in many bacterial species they are the principal manganese import system. Nramps are metal-proton symporters, using a pH gradient to drive the co- transport of divalent metals and protons. The overall goal is to determine the molecular mechanism of metal ion-proton symport by the Nramp family of proteins through biochemical, computational, and structural studies of bacterial Nramp proteins. We recently determined the crystal structure of a bacterial Nramp homolog, which serves as a scaffold for generating hypotheses and interpreting data. We have developed a range of in vivo and in vitro activity assays, which we will use to define how metal selectivity is encoded in the sequences of Nramp family transporters. We will also use these assays to understand how protons and protonation events influence the transport cycle. We will study the conformation changes during metal transport using biochemical approaches, engineer constructs that stabilize particular conformational states for high-resolution structure determination by x-ray crystallography, and molecular dynamic simulations in various protonation states to model dynamics produced by protonation or deprotonation events. Our overall goal is to establish an atomic- resolution model of the proton-coupled metal ion transport cycle. While Nramps are part of the well-characterized LeuT-fold superfamily of transporters, they are unusual because they are neither sodium-coupled symporters nor antiporters. Our proposed research focuses on several of Nramps’ unique features and will thus expand our knowledge of the mechanistic diversity enabled by the LeuT fold. Both bacterial and mammalian Nramp proteins have an impact on human health. Bacterial Nramps increase pathogenicity by facilitating the uptake of essential divalent metals. Human Nramps are particularly important in immunity to intracellular pathogens, liver and blood homeostasis, and brain function. Nramps have been implicated in numerous pathologic conditions including autoimmune diseases, anemia and Parkinson disease. The proposed basic research will have a major impact on the field by providing sorely needed mechanistic information on the biomedically relevant class of Nramp transporters. These structural and mechanistic insights into metal ion transport by Nramp proteins can eventually contribute to the development of drugs and therapeutic strategies to treat disorders resulting from imbalances in metal ion homeostasis.

研究摘要 铁和锰等金属对于包括氧气在内的许多物理过程至关重要 运输和能源代谢。但是这些金属的不足是有毒的，它们的身体水平 因此受到严格的调节。 NRAMP（自然抗性相关的巨噬细胞蛋白）是膜 将二价金属离子导入细胞的转运蛋白。 NRAMP对于两种二价金属吸收都很重要 金属将金属进口到细胞质中。因此，nramp是维持的关键因素 二价金属的稳态，尤其是铁和锰。 NRAMP蛋白从细菌到人都保守，在许多细菌中，它们是 主要锰进口系统。 NRAMP是金属 - 普罗替型对甲，使用pH梯度来驱动共同 二价金属和质子的运输。总体目标是确定金属的分子机制 蛋白质的NRAMP家族通过生化，计算和结构研究由NRAMP家族的离子 - 普罗顿交易 细菌NRAMP蛋白。我们最近确定了细菌同源物的晶体结构，该结构 作为产生假设和解释数据的脚手架。我们已经开发了一系列体内 和体外活性评估，我们将用来定义金属选择性如何以 NRAMP家庭转运蛋白。我们还将使用这些测定法来了解质子和质子化事件 影响运输周期。我们将使用生化的金属运输过程中的会议变化 方法，工程师的构造，稳定高分辨率结构的特定概念状态 通过X射线晶体学确定各种质子化状态的分子动态模拟 由质子化或去质子化事件产生的模型动力学。我们的总体目标是建立一个原子 - 质子耦合金属离子传输周期的分辨率模型。 虽然NRAMP是转运蛋白特征良好的Leut折叠超级家族的一部分，但它们是不寻常的 因为它们既不是钠耦合的共菌也不是抗植物。我们提出的研究重点 NRAMP的一些独特功能将扩大我们对由机械多样性的了解 Leut折叠。细菌和哺乳动物NRAMP蛋白都对人类健康有影响。细菌 NRAMP通过促进基本二价金属的吸收来增加致病性。人类nramp是 在细胞内病原体，肝脏和血液稳态以及大脑功能的免疫学中尤为重要。 在包括自身免疫性疾病，贫血和 帕金森病。拟议的基础研究将对该领域产生重大影响 关于生物医学相关类别的NRAMP转运蛋白类所需的机械信息。这些结构和 NRAMP蛋白对金属离子传输的机械洞察有时会有助于发展 药物和治疗策略治疗因金属离子稳态失衡而导致的疾病。