Mechanism of Divalent Metal Transport by Nramp-Family Transporters

项目总结-Nramp家族转运蛋白转运二价金属的机制 背景：铁和锰等金属对许多生理过程至关重要 包括氧气运输和能量代谢。但这些离子的过量或不足会导致 健康问题--包括贫血、血色病和免疫或神经系统疾病-- 并且它们的生理水平因此受到严格调节。Nramps（自然电阻相关 巨噬细胞蛋白）是将金属离子和质子输入细胞的共转运体，因此被 对维持过渡金属的体内平衡至关重要。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 用于过渡金属离子底物的位点和由极性残基网络形成的质子通路 在蛋白质支架中。此外，有证据表明，遥远的Nramp同源物 金属结合序列基序的变化和运输其他金属，如Al 3+和Mg 2+。 建议的研究：我们的目标是双重的：（i）发展原子水平的生物物理理解 大多数真核生物和细菌Nramp共有的典型机制特征;以及（ii） 将这些特征与更远的Nramp样同源物中的特征进行对比。我们将联合收割机序列 生物信息学和其他计算方法与结构和生物化学分析。在 目的1，我们使用一个成熟的细菌Nramp转运蛋白研究其典型特征， 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发生化学偶联 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， 我们研究了具有非典型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）质子运输，（2b） 金属选择性和（2c）金属配位几何形状。我们的两个目标协同作用，以提供- 深入的生物物理机制和这个重要的转运蛋白家族的广阔前景。 影响：细菌和哺乳动物的Nramp都会影响人类健康。在细菌中，Nramp有助于 浮游微生物获得必需的过渡金属并促进病原体的定植。 人Nramp对于对细胞内病原体的免疫、肝脏和血液稳态是必需的， 和大脑功能。Nramps对金属离子转运的这项研究提供了生物化学和 生物物理学基础，必要的解释他们的基本作用，在金属稳态的细胞 有机体水平。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、血色病和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.