Mechanism of Divalent Metal Transport by Nramp-Family Transporters

项目摘要 - Nramp 家族转运蛋白的二价金属转运机制 背景：铁和锰等金属对于许多生理过程至关重要 包括氧气运输和能量代谢。但这些离子过多或缺乏会导致 健康问题——包括贫血、血色素沉着症和免疫或神经系统疾病—— 因此它们的生理水平受到严格调节。 Nramps（自然抵抗力相关 巨噬细胞蛋白）是将金属离子和质子输入细胞的同向转运蛋白，因此 对于维持过渡金属稳态至关重要。然而，耦合机制 金属离子和质子之间的关系尚不清楚。细菌 Nramp 的结构揭示了结合 过渡金属离子底物的位点和由极性残基网络形成的质子路径 在蛋白质支架中。此外，越来越多的证据表明，遥远的 Nramp 同源物已经 金属结合序列基序的变化并运输其他金属，如 Al3 和 Mg2 。 拟议的研究：我们的目标有两个：（i）发展原子级生物物理理解 大多数真核生物和细菌 Nramps 共有的典型机制特征； (二) 将这些特征与更遥远的类似 Nramp 的同源物进行对比。我们组合序列 生物信息学和其他具有结构和生化分析的计算方法。在 目标 1，我们使用成熟的细菌研究 Nramp 转运蛋白的典型特征 Nramp 模型系统。我们将确定 (1a) 金属离子配位几何形状和亲和力 选择性决定因素，(1b) 质子传输是否与金属热力学耦合 运输，以及 (1c) 蛋白质的质子化状态如何改变构象动力学。在目标 2 中， 我们研究具有非规范金属结合和质子途径的不同 Nramp 同系物 序列基序。我们研究这些序列变化如何影响 (2a) 质子传输，(2b) 金属选择性，以及（2c）金属配位几何形状。我们的两个目标相辅相成，提供in- 深入的生物物理机制和这个重要的转运蛋白家族的广阔视角。 影响：细菌和哺乳动物 Nramp 都会影响人类健康。在细菌中，Nramps 有帮助 共生微生物获得必需的过渡金属并促进病原体的定殖。 人类 Nramp 对于细胞内病原体的免疫、肝脏和血液稳态至关重要， 和大脑功能。这项关于 Nramps 金属离子转运的研究提供了生化和 解释它们在细胞金属稳态中的重要作用所必需的生物物理基础 和组织水平。这些知识可以为金属相关疾病提供更好的治疗方法 包括贫血、血色素沉着症以及许多免疫和神经系统疾病。

PROJECT SUMMARY - Mechanism of Divalent Metal Transport by Nramp-Family Transporters Background: Metals like iron and manganese are essential to many physiological processes including oxygen transport and energy metabolism. But excess or deficiency of these ions leads to health issues—including anemia, hemochromatosis and immune or neurological disorders— and their physiological levels are thus tightly regulated. Nramps (natural resistance-associated macrophage proteins) are symporters that import metal ions and protons into cells, and thus are crucial to maintaining transition metal homeostasis. However, the mechanism of coupling between metal ions and protons is unclear. Structures of bacterial Nramps revealed the binding site for the transition metal ion substrate and a proton pathway formed by a polar residue network in the protein scaffold. Furthermore, evidence is emerging that distant Nramp homologs have variations of the metal-binding sequence motifs and transport other metals like Al3+ and Mg2+. Proposed Research: Our goal is two-fold: (i) develop an atomic-level biophysical understanding of the canonical mechanistic features shared by most eukaryotic and bacterial Nramps; and (ii) contrast these features to those in more distant Nramp-like homologs. We combine sequence bioinformatics and other computational approaches with structural and biochemical analyses. In Aim 1, we investigate canonical features of Nramp transporters using a well-established bacterial Nramp model system. We will determine (1a) the metal ion coordination geometry and affinity and selectivity determinants, (1b) whether proton transport is thermodynamically coupled to metal transport, and (1c) how protonation states of the protein alter conformational dynamics. In Aim 2, we investigate divergent Nramp homologs with noncanonical metal-binding and proton-pathway sequence motifs. We examine how these sequence changes affect (2a) proton transport, (2b) metal selectivity, and (2c) metal coordination geometries. Our two aims synergize to provide in- depth biophysical mechanisms and a broad perspective of this important family of transporters. Impact: Both bacterial and mammalian Nramps impact human health. In bacteria, Nramps help commensal microbes acquire essential transition metals and promote colonization by pathogens. Human Nramps are essential for immunity to intracellular pathogens, liver and blood homeostasis, and brain function. This research on metal ion transport by Nramps provides the biochemical and biophysical grounding necessary to explain their essential role in metal homeostasis at the cellular and organismal level. This knowledge could lead to better therapies for metal-related diseases including anemia, hemochromatosis, and many immune and neurological disorders.