Mechanism of Divalent Metal Transport by NRamp-Family Transporters

研究总结 铁和锰等金属是包括氧气在内的许多生理过程所必需的。 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 因此受到严格的监管。Nramps(自然抵抗相关巨噬细胞蛋白)是一种膜蛋白 将二价金属离子输入细胞的转运体。Nramp对于两种二价金属的吸收都很重要 通过饮食和细胞将金属输入胞质。因此，NRAMP是维护 二价金属，特别是铁和锰的动态平衡。 Nramp蛋白在细菌和人类之间是保守的，在许多细菌物种中，它们是 主要的锰矿进口制度。Nramps是金属-质子共转运体，使用pH梯度来驱动co- 二价金属和质子的运输。总体目标是确定金属的分子机制。 Nramp蛋白质家族通过生化、计算和结构研究实现离子-质子共生 细菌Nramp蛋白。我们最近确定了一种细菌Nramp同源物的晶体结构，它 作为生成假设和解释数据的脚手架。我们已经开发出一系列体内的 以及体外活性分析，我们将用它来定义金属选择性是如何在以下序列中编码的 Nramp家族的运输机。我们还将使用这些分析来了解质子化和质子化事件 影响运输周期。我们将利用生物化学来研究金属运输过程中的构象变化 方法，工程构造，稳定高分辨率结构的特定构象状态 用X射线结晶学和分子动力学模拟在不同质子化状态下测定 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- 质子耦合金属离子输运循环的解析模型。 虽然Nramps是典型的Leut-Fold运输机超家族的一部分，但它们是不寻常的 因为它们既不是钠偶联的同向转运体，也不是反向转运体。我们建议的研究重点是 Nramps的几个独特功能，从而将扩展我们对Nramps实现的机械多样性的知识 Leut折叠店。细菌和哺乳动物的Nramp蛋白都对人类健康有影响。细菌 Nramps通过促进必需的二价金属的吸收来增加致病性。人类的坡道是 在对细胞内病原体的免疫、肝脏和血液的动态平衡以及大脑功能方面尤为重要。 Nramps与许多病理情况有关，包括自身免疫性疾病、贫血和 帕金森氏症。拟议的基础研究将对该领域产生重大影响，因为它提供了 需要关于生物医学相关类Nramp转运体的机械信息。这些结构和 对Nramp蛋白对金属离子运输的机械洞察最终可以促进 治疗金属离子动态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.