Structure and Function of Copper Transport ATPases

铁、铜、锌等金属微量营养素在蛋白质中起着基本的催化和结构作用。适当的金属水平是通过特定的伴侣和跨膜转运蛋白的协调作用来实现的。本项目的目标是了解普遍存在的P1B型ATPase参与重金属跨膜运输的基本生物化学。碱金属(Na+、K+、Ca2+)在细胞环境中是游离的(水合的)。因此，它们可逆地与通道、联合传送器和泵相互作用并由其运输。相反，重金属与具有较高缔合常数的伴侣/螯合分子结合。该项目旨在了解重金属是如何输送到转运体的，它们是如何与可能高于其伴侣的亲和力协调的，以及它们随后是如何从转运体中释放出来的。在这些研究中，黄褐古球藻将被用作铜-ATPase的模型。将进行实验，以测试铜绿假单胞菌的铜离子伴侣蛋白CopZ是否与CopA相互作用，从而将铜离子输送到跨膜金属结合部位。随着原子配位和配位氨基酸侧链的排列，金属与跨膜位置结合的化学计量学将被建立。该项目还将研究通过金属与细胞质结构域结合来调节变构的结构机制。最后，该项目的第四个目标是建立一个完整的铜离子转运ATPase的原子分辨结构。这将极大地提高对重金属迁移机制的理解。更广泛的影响：该项目将有助于理解管理金属运输器的金属结合、转移和释放机制的原则。这项研究将有助于正在进行的教育活动，包括本科教学、课程开发和暑期研究。研究生和本科生是实验室的核心，他们将完成这个项目的主要部分。这项工作还将影响我们机构与K-12项目的密切合作。这包括指导高中生在实验室工作，以及参与高中教师培训。

Metal micronutrients, Fe, Cu, Zn, etc., play fundamental catalytic and structural functions in proteins. Proper metal levels are achieved by the coordinate action of specific chaperones and transmembrane transporters. The goal of this project is to understand the fundamental biochemistry involved in transmembrane heavy metal transport by ubiquitous P1B-type ATPases. Alkali metals (Na+, K+, Ca2+) are free (hydrated) in the cellular milieus. As such, they reversibly interact with and are transported by channels, co-transporters, and pumps. In contrast, heavy metals are bound to chaperones/chelating molecules with high association constants. This project aims to understand how heavy metals are delivered to their transporters, how they are coordinated with affinities likely higher than that of their chaperones, and how they are subsequently released from the transporter. In these studies, Archaeoglobus fulgidus CopA will be used as a model Cu+-ATPase. Experiments will be performed to test whether CopZ, the A. fulgidus Cu+ chaperone, interacts with CopA delivering Cu+ to transmembrane metal binding sites. The stoichiometry of metal binding to transmembrane sites will be established along with the atomic coordination and the arrangement of coordinating amino acid side chains. The project will also examine the structural mechanism of allosteric regulation by metal binding to cytoplasmic domains. Finally, the fourth goal of the project is to establish the complete atomic resolution structure of a Cu+ transporting ATPase. This would greatly enhance understanding the mechanism of heavy metal transport. Broader Impact: The project will contribute to understanding the principles governing metal binding, translocation and release mechanisms of metal transporters. The research will contribute to ongoing educational activities including undergraduate teaching, curriculum development and summer research. Graduate and undergraduate students form the core of the laboratory and will perform major parts of this project. This work will also impact a close collaboration with K-12 programs at our institution. This ranges from guiding high school students working in the lab to participation in high school teachers training.