Mechanism of Divalent Metal Transport by Nramp-Family Transporters

项目总结-Nramp家族转运体运输二价金属的机制 背景：铁和锰等金属是许多生理过程所必需的 包括氧气运输和能量代谢。但这些离子的过量或不足都会导致 到健康问题--包括贫血、血色沉着症和免疫或神经疾病-- 因此，它们的生理水平受到严格的调控。Nramps(自然抗性相关 巨噬细胞蛋白)是将金属离子和质子输入细胞的代谢物，因此 对维持过渡金属动态平衡至关重要。然而，耦合的机制 金属离子和质子之间的关系尚不清楚。细菌Nramp的结构揭示了这种结合 过渡金属离子底物的位置和由极性残基网络形成的质子路径 在蛋白质支架上。此外，正在出现的证据表明，遥远的Nramp同源物 金属结合序列基序的变异并运输其他金属，如Al3和Mg2。 拟议研究：我们的目标有两个：(I)发展原子水平的生物物理理解 大多数真核生物和细菌Nramps共有的典型机制特征；及(Ii) 将这些特征与更远的Nramp类同源基因中的特征进行比较。我们结合了序列 生物信息学和其他具有结构和生化分析的计算方法。在……里面 目的1，我们使用一种成熟的细菌来研究Nramp转运蛋白的典型特征 Nramp模型系统。我们将确定(1a)金属离子的配位几何构型和亲和力 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， 我们研究了具有非正则金属结合和质子途径的发散Nramp同系物 序列基序。我们研究了这些序列变化如何影响(2a)质子传输，(2b) 金属选择性和(2c)金属配位几何构型。我们的两个目标协同提供- 深入的生物物理机制和广阔的前景这一重要的转运体家族。 影响：细菌和哺乳动物的Nramps都会影响人类健康。在细菌中，Nramps有助于 共生微生物获得必需的过渡金属，并促进病原体的定居。 人类Nramps对于细胞内病原体的免疫、肝脏和血液的动态平衡是必不可少的， 和大脑功能。Nramps对金属离子输运的研究为生物化学和生物化学研究提供了依据。 解释它们在细胞内金属动态平衡中的重要作用所需的生物物理基础 和生物体水平。这一知识可能会为金属相关疾病带来更好的治疗方法 包括贫血，血色沉着症，以及许多免疫和神经疾病。

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.