Mechanism of Divalent Metal Transport by NRamp-Family Transporters

研究综述 铁和锰等金属对包括氧在内的许多生理过程都是必不可少的 运输和能量代谢。但过量的这些金属是有毒的，它们的生理水平 因此受到严格管制。NRAMP（天然抗性相关巨噬细胞蛋白）是膜 将二价金属离子输入细胞的转运蛋白。NRAMP对于二价金属的吸收都是重要的， 饮食和金属进入细胞质的细胞进口。因此，NRAMP是维持 二价金属，特别是铁和锰的体内平衡。 Nramp蛋白从细菌到人类都是保守的，在许多细菌物种中，它们是 主要锰进口系统。Nramp是金属-质子同向转运体，使用pH梯度来驱动共- 二价金属和质子的传输。总体目标是确定金属的分子机理 通过生物化学、计算和结构研究，Nramp蛋白质家族的离子-质子共转运 细菌Nramp蛋白质。我们最近确定了细菌Nramp同系物的晶体结构， 充当生成假设和解释数据的支架。我们已经开发了一系列的体内 和体外活性测定，我们将使用这些测定来确定金属选择性如何在以下序列中编码： Nramp家族运输机。我们还将使用这些分析来了解质子和质子化事件 影响运输周期。我们将利用生物化学方法研究金属转运过程中的构象变化 方法，工程师构建稳定特定构象状态的高分辨率结构 通过X射线晶体学测定，以及在各种质子化状态下的分子动力学模拟， 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循环的分辨率模型。 虽然Nramps是转运蛋白LeuT折叠超家族的一部分，但它们是不寻常的 因为它们既不是钠偶联的同向转运蛋白，也不是反向转运蛋白。我们的研究重点是 Nramps的几个独特的功能，从而将扩大我们的知识的机械多样性，使之成为可能， LeuT折叠。细菌和哺乳动物的Nramp蛋白都对人类健康有影响。细菌 NRAMP通过促进必需的二价金属的摄取来增加致病性。人类Nramp是 在对细胞内病原体的免疫、肝脏和血液稳态以及脑功能中特别重要。 NRAMP已经涉及许多病理状况，包括自身免疫性疾病、贫血和免疫性疾病。 帕金森病。拟议中的基础研究将对该领域产生重大影响， 需要关于Nramp转运蛋白的生物医学相关类别的机械信息。这些结构和 Nramp蛋白对金属离子转运的机制见解最终有助于开发 用于治疗由金属离子体内平衡失衡引起的疾病的药物和治疗策略。

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.