Dynamics and Interactions of Metal-Binding Domains in the Function of Wilson Disease Protein.

Wilson disease protein (ATP7B) is a P-type ATPase that catalyzes the transport of copper across cell membranes and is critical for maintaining copper homeostasis. ATP7B contains an N-terminal metal binding module that is found only in heavy metal transporters. The N-terminal region is composed of six metal-binding domains (MBD1-6) joined together by flexible linkers. Each MBD is approximately 70 amino acids and contains the signature motif, CXXC, with one Cu(I) binding between two cysteines. The structures of the individual MBDs have been solved, however, the dynamics and arrangement of the individual MBDs in the full-length protein were unknown. Gaining insights into the conformation and dynamics of this unique region helps us understand how the enzymatic and trafficking activity of ATP7B is regulated. In our study, we used recombinant single domain camelid antibodies, also known as nanobodies, to reveal functionally relevant transient interactions within a distinct group of MBDs. We identified new transient interactions between metal-binding domains 1, 2, and 3 and provided independent evidence for structurally and functionally distinct roles for MBD5-6. We demonstrated that nanobodies were used as effective probes for investigating molecular dynamics of multi-domain proteins. Overexpression of ATP7B is associated with tumor resistance to cisplatin during chemotherapy. Cisplatin kills cancer cells by reacting with DNA, which prevents proper DNA transcription and replication. ATP7B was proposed to actively transport cisplatin out of the cell, however, low transport rates in physiological pH and high substrate specificity for copper suggests that cisplatin detoxification by active extrusion is unlikely. Rather, we proposed that the metal-binding domains in ATP7B functioned as a high-affinity reservoir by trapping cisplatin and then releasing it to other potential terminal acceptors in a non-toxic form. We used NMR spectroscopy to characterize cisplatin binding to metal-binding domain 2 (MBD2) of ATP7B. We demonstrated that platinum is transferred from Atox1 to MBD2 in a mechanism that parallels the copper-transport pathway in the cell, which suggested a possible route for cisplatin detoxification. Platinum was subsequently released to other terminal acceptors such as glutathione and glutaredoxin, which demonstrated that the metal-binding domains may not be the final destination for cisplatin