Principles of selectivity and translocation in transition metal transporter

摘要 过渡金属是生命所必需的微量元素，在生物化学过程中起着关键作用。第一排 过渡金属，如铁、铜和锌，发挥着基本的催化、结构和信号功能。 它们的浓度受到严格控制，以满足必要的细胞需求，而不会产生毒性 级别。另一方面，非必需的第二/第三排过渡金属络合物对细胞和 被用作治疗分子。在所有细胞中控制金属浓度的把关角色是被介导的。 通过跨膜转运蛋白调节跨细胞的金属摄取和排泄 膜。金属选择性的基本原理和通过这些物质传输的分子机制 纳米机器仍然难以捉摸。 这项MIRA应用的目标是研究主要的活性过渡金属泵和溶质载流子(SLC)和 将侧重于：(1)研究第一、第二和第三排过渡金属的金属选择性原则 和(Ii)它们的金属配位化学；(Iii)确定金属移位途径；(Iv)解决 分子水平上能量传递过程的机制。 我的实验室已经开发出一种综合的化学、生物物理和结构方法来确定 金属的选择和运输发生在不同的金属运输者家族中。有了这一多学科战略， 我们将以已知的和新的转运蛋白家族为目标，这些家族涉及金属动态平衡和疾病进展； 具体地说：1)P1B型ATPase，控制人类细胞内铜水平的主要活性转运体， 以及调节病原菌中铜等过渡金属的浓度；2)TMEM205，a 可能参与铜排出和抗癌铂复合体的新型人类转运蛋白 运输和抗性；3)IroT转运体，可能是铁调节的溶质载体，负责铁(II) 致病原核生物的获取和毒力。 我们将结合生物物理、光谱和结构研究纯化的洗涤剂增溶的转运蛋白。 与蛋白质脂质体中的特征有关，其中转运体嵌入天然的类脂双层中。通过 包裹在蛋白脂质体中的金属依赖的荧光螯合剂、二次离子传感器和 膜电位的探针，我们将开发一种体外工具，用于实时监测底物转运。 一种膜环境。该平台建立了一个创新的框架，以解决i)金属衬底 选择性，II)共传输离子的性质，III)它们的相对化学计量比，IV)电生性质，以及 (V)膜电位在金属催化移位中的作用。 该项目的目标是生物无机化学中被忽视的一个方面，即对原理的理解。 控制金属跨膜转移。除了遮蔽基本的分子机制之外 在金属运输方面，对新靶子的研究将对翻译发现产生重大影响。

Abstract Transition metals are essential trace elements for life, playing pivotal roles in biochemical processes. First-row transition metals, such as iron, copper, and zinc, play fundamental catalytic, structural and signaling functions. Their concentrations are tightly regulated to meet indispensable cellular requirements without reaching toxic levels. On the other hand, non-essential second-/third-row transition metal complexes are toxic to cells and exploited as therapeutic molecules. A gatekeeper role in controlling metal concentrations in all cells is mediated by transmembrane transporters that regulate the vectorial metal uptake and extrusion across cellular membranes. The principles underlying metal selectivity and molecular mechanism of transport by these nanomachines remain elusive. This MIRA application targets the study of primary active transition metal pumps and solute carriers (SLC) and will focus on: (i) investigating the principles of metal selectivity for first, second- and third- row transition metals and (ii) their metal coordination chemistry; (iii) determining the metal translocation pathway; (iv) addressing the mechanisms of energy transduction processes at a molecular level. My laboratory has developed an integrated chemical, biophysical, and structural approach to determine how metal selection and transport occurs in different metal transporter families. With this multidisciplinary strategy, we will target known and novel transporter families involved in metal homeostasis and in disease progression; specifically: 1) P1B-type ATPases, primary active transporters controlling intracellular copper levels in humans, and modulating the concentrations of copper and other transition metals in pathogenic bacteria; 2) TMEM205, a novel human transporter potentially involved in copper extrusion and responsible for anti-cancer Pt-complexes transport and resistance; 3) IroT transporters, putative iron-regulated solute carriers responsible for iron(II) acquisition and virulence in pathogenic prokaryotes. We will couple biophysical, spectroscopic, and structural studies on purified, detergent-solubilized transporters to the characterization in proteoliposomes, where the transporters are embedded in a native-like lipid bilayer. By encapsulating in the proteoliposomes metal-dependent fluorescence chelators, sensors for secondary ions, and probes for membrane potential, we will develop an in vitro tool for monitoring real-time substrate translocation in a membrane environment. This platform establishes an innovative framework to address i) the metal substrate selectivity, ii) the nature of cotransported ions, iii) their relative stoichiometry, iv) the electrogenic properties, and v) the role of membrane potential on catalytic metal translocation. The project targets a neglected aspect of bioinorganic chemistry towards the understanding of the principles controlling metal translocation across membranes. Besides shading light on the basic molecular mechanisms governing metal transport, the study of novel targets will have a major impact on translational discoveries.