Structure-Function of Heavy Metal Transport CPx-ATPases

本项目的目的是了解重金属转运CPX-ATPase的作用机制。这些P型ATPase通过生物膜转运重金属(铜、银、锌、钴、镉、铅等离子)。在所有生命王国中都发现了这些关键酶，它们参与了微量营养素的金属动态平衡。CPX-ATPase的转运化学计量学、金属离子专一性的决定因素和调控机制尚不清楚。该项目旨在确定涉及离子选择性和活性调节的关键结构特征。在原核生物中发现的更简单的CPX-ATPase将被用作这些研究的模型：来自黄曲霉的Cu-ATPase，来自A.pernix的Co-ATPase，以及来自流感嗜血杆菌的锌-ATPase。黄曲霉和Pernix是极端嗜热的古生菌。将进行实验，以测试CPX-ATPase跨膜区的保守序列是否决定离子特异性。CPX-ATPase除了具有跨膜CPX位点外，还具有胞质N端金属结合域。金属结合域在酶动力学中可能的调节作用将被研究。该项目还将涉及测试金属伴侣分子是否将金属运送到运输地点。该项目的第四个目标是利用X射线结晶学确定CPX-ATPase的结构。CPX-ATPase在原子分辨率下的结构将极大地提高对重金属转运机制的理解。更广泛的影响：该项目将有助于理解重金属的动态平衡。在金属选择性方面获得的洞察力可能有助于设计金属生物修复工艺和管理动植物矿物质营养。这项研究还将有助于正在进行的教育活动(本科教学、课程开发和暑期研究)。此外，本科生和研究生将完成这个项目的主要部分。

The goal of this project is to understand the functional mechanism of heavy metal transport CPx-ATPases. These P-type ATPases transport heavy metals (ions of Cu, Ag, Zn, Co, Cd, Pb) across biomembranes. Found in all life kingdoms, these key enzymes are involved in micronutrient metal homeostasis. The transport stoichiometry, the determinants of metal ion specificity, and the regulatory mechanisms of CPx-ATPases are not known. This project aims to identify key structural features involved in ion selectivity and activity regulation. Simpler CPx-ATPases found in procaryotes will be used as models in these studies: Cu-ATPases from A. fulgidus, a Co-ATPase from A. pernix, and a Zn-ATPase from H. influenzae. A. fulgidus and A. pernix are extremophilic archaea. Experiments will be performed to test whether conserved sequences in the transmembrane region of CPx-ATPases determine ion specificity. CPx-ATPases have cytoplasmic N-terminal metal-binding domains, in addition to the transmembrane CPx site. The possible regulatory role of the metal-binding domain in enzyme kinetics will be studied. This project will also involve testing whether a metal chaperone molecule delivers the metal to the transport site. The fourth goal of this project is to determine the structure of a CPx-ATPase using X-ray crystallography. The structure of CPx-ATPases at atomic resolution would greatly enhance understanding the mechanism of heavy metal transport.Broader Impact: The project will contribute to an understanding of heavy metal homeostasis. Insight gained in metal selectivity might facilitate the design of metal bioremediation processes and the management of plant and animal mineral nutrition. This research will also contribute to ongoing educational activities (undergraduate teaching, curriculum development and summer research). Moreover, undergraduate and graduate students will perform major parts of this project.