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助因子，但过量的铜对细胞具有高度毒性，并且铜在人体细胞中的获取、分布和去除受到严格控制。铜在细胞中的运输过程是复杂的，目前还没有很好地了解。我们研究的长期目标是阐明人体组织中铜转运的分子机制。细胞内铜代谢的关键酶是两种结构相似的atp驱动的膜转运蛋白ATP7B和ATP7A，这是本研究计划的重点。ATP7B的酶活性和细胞内定位受铜的调控。在细胞中，铜由伴侣蛋白Atox1传递到ATP7B的六个细胞质金属结合域（MBD1-6）链上。MBD1-6的构象、分子内铜转移的后续步骤以及铜调控ATP7B的机制尚不清楚。我们的中心假设是，相互作用和MBD链的三维折叠中的铜依赖变化触发ATP7B从反式高尔基网络重新定位到胞质膜囊泡和质膜，并激活铜跨细胞膜和细胞外的运输。利用高分辨率核磁共振、小角度x射线散射（SAXS）和生化方法，我们将确定MBD1-6的构象和动力学如何调节MBD1-6的活性和细胞内定位。这项工作将对真核多结构域蛋白生物学调控的基本原理提供新的认识，并促进我们对人体细胞中金属转运过程的认识。

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.