Principles of selectivity and translocation in transition metal transporter

摘要 过渡金属是生命必需的微量元素，在生物化学过程中起着关键作用。First-row 过渡金属如铁、铜和锌起着基本的催化、结构和信号功能。 它们的浓度受到严格控制，以满足不可或缺的细胞需求，而不会产生毒性。 程度.另一方面，非必需的第二/第三行过渡金属络合物对细胞有毒， 被用作治疗分子。在所有细胞中控制金属浓度的看门人作用是介导的 通过跨膜转运蛋白调节跨细胞的载体金属摄取和排出， 膜。这些分子的金属选择性和传输机制的基本原理 纳米机器仍然难以捉摸。 该MIRA应用的目标是研究初级活性过渡金属泵和溶质载体（SLC）， 主要研究方向为：（1）第一、第二、第三行过渡金属的选择性原理 和（ii）它们的金属配位化学;（iii）确定金属易位途径;（iv）解决 分子水平上的能量转换过程的机制。 我的实验室开发了一种综合化学，生物物理和结构方法来确定如何 金属选择和转运发生在不同的金属转运蛋白家族中。通过这一多学科战略， 我们将靶向参与金属稳态和疾病进展的已知和新的转运蛋白家族; 具体地说：1）P1 B型ATP酶，在人体中控制细胞内铜水平的主要活性转运蛋白， 以及调节病原菌中铜和其它过渡金属的浓度; 2）TMEM 205，a 一种可能参与铜外排并负责抗癌铂络合物的新型人类转运蛋白 3）IroT转运蛋白，推定的铁调节溶质载体，负责铁（II） 致病原核生物中的获得和毒力。 我们将结合生物物理学、光谱学和结构学对纯化的、洗涤剂溶解的转运蛋白进行研究 蛋白脂质体中的特征，其中转运蛋白嵌入天然样脂质双层中。通过 在蛋白脂质体中包封金属依赖性荧光螯合剂、二次离子传感器，和 探针的膜电位，我们将开发一种体外工具，用于监测实时底物易位， 膜环境。该平台建立了一个创新的框架，以解决i）金属基板 选择性，ii）共转运离子的性质，iii）它们的相对化学计量，iv）产电性质，和 v）膜电位在催化金属迁移中的作用。 该项目针对生物无机化学中被忽视的一个方面，以了解其原理 控制金属跨膜迁移。除了在基本的分子机制上遮遮掩掩 新靶点的研究将对转化发现产生重大影响。

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.