Structure-Function of Heavy Metal Transport CPx-ATPases

本项目旨在了解重金属转运cpx - atp酶的功能机制。这些p型atp酶通过生物膜运输重金属（Cu、Ag、Zn、Co、Cd、Pb离子）。这些关键酶存在于所有生命王国中，参与微量营养素金属的体内平衡。转运化学计量学、金属离子特异性的决定因素以及cpx - atp酶的调节机制尚不清楚。该项目旨在确定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 - atp酶将被用作这些研究的模型：来自a . fulgidus的cu - atp酶，来自a . pernix的co - atp酶和来自流感嗜血杆菌的zn - atp酶。A. fulgidus和A. pernix是嗜极古菌。实验将测试cpx - atp酶跨膜区域的保守序列是否决定离子特异性。CPx- atp酶除了具有跨膜CPx位点外，还具有胞质n端金属结合结构域。将研究金属结合结构域在酶动力学中的可能调节作用。该项目还将包括测试金属伴侣分子是否将金属运送到运输部位。该项目的第四个目标是利用x射线晶体学确定cpx - atp酶的结构。cpx - atp酶在原子分辨率上的结构将大大提高对重金属转运机制的认识。更广泛的影响：该项目将有助于了解重金属的动态平衡。在金属选择性方面获得的见解可能有助于金属生物修复过程的设计和植物和动物矿物质营养的管理。这项研究也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，本科生和研究生将执行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.