Molecular Mechanism of Regulation of the Human Copper Transporter ATP7B

铜是许多生物过程中必不可少的辅因子，但过量的铜对细胞具有高度毒性，并且铜从人体细胞中的获取，分布和去除受到严格控制。铜在细胞中的转运过程是复杂的，目前还没有很好的理解。本研究的长期目标是阐明铜在人体组织中转运的分子机制。细胞中铜代谢的关键酶是两种结构相似的ATP驱动的膜转运蛋白，即ATP 7 B和ATP 7A。ATP 7 B的酶活性和细胞内定位受铜的调节。在细胞中，由伴侣蛋白Atox 1递送的铜被转移到ATP 7 B的六个胞质金属结合结构域（MBD 1 -6）的链上。MBD 1 -6的构象、随后的分子内铜转移步骤以及铜对ATP 7 B的调节机制尚不清楚。我们的中心假设是，铜依赖性的变化的相互作用和三维折叠的MBD链触发重新定位的ATP 7 B从trans-Golgi网络的胞质膜囊泡和质膜，并激活铜跨细胞膜和细胞外的运输。使用高分辨率NMR，小角X射线散射（SAXS）和生物化学方法，我们将确定MBD 1 -6的构象和动力学如何调节MBD 1 -6的活性和细胞内定位。这项工作将提供新的见解的多域真核生物蛋白质的生物调控的基本原则，并推进我们的知识在人类细胞中的金属运输过程。

Copper is an essential cofactor in many biological processes, yet excess of copper is highly toxic for the cells, and acquisition, distribution and removal of copper from the human cells is tightly controlled. Copper transport processes in the cell are complex and not well understood at present. The long term goal of our research is to elucidate the molecular mechanism of copper transport in human tissues. The key enzymes in copper metabolism in the cell are two structurally similar ATP-driven membrane transporters, ATP7B, which is the focus of this research proposal, and ATP7A. Enzymatic activity and intracellular localization of ATP7B are regulated by copper. In the cell, copper, delivered by a chaperone protein Atox1, is transferred to the chain of six cytosolic metal binding domains (MBD1-6) of ATP7B. Conformation of MBD1-6, the subsequent steps of intramolecular copper transfer, and the mechanism of ATP7B regulation by copper are unknown. Our central hypothesis is that copper-dependent changes in the interactions and the three-dimensional fold of the MBD chain trigger relocalization of ATP7B from the trans-Golgi network to the cytosolic membrane vesicles and plasma membrane, and activate copper transport across the cell membranes and out of the cell. Using high-resolution NMR, small-angle X-ray scattering (SAXS) and biochemical methods, we will determine how the conformation and dynamics of MBD1-6 regulate activity and intracellular localization of MBD1-6. This work will provide new insight into the fundamental principles of biological regulation of multidomain eukaryotic proteins, and advance our knowledge of metal transport processes in the human cells.