Structural organization and mechanisms of cooperativity in the N-Terminal domains of human CU-ATpases.
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- Copper; Adenosine triphosphatase; Oxidation-reduction reaction; Proteins -- Structure; Molecular chaperones; Wilson disease protein; Menkes disease protein; ATOX1 protein; human; Adenosine Triphosphatases; Oxidation-Reduction; Protein Structure; Quaternary; Molecular Chaperones; Wilson disease protein; Menkes disease protein; ATOX1 protein, human; Protein Structure, Quaternary
thesis / dissertation description
Copper is an essential trace element used as a cofactor by numerous enzymes in the catalysis of electron transfer reactions. The reactivity of copper necessitates strict homeostatic mechanisms to maintain necessary concentrations while also preventing toxicity. The disruption of copper homeostasis can be seen in the severe genetic diseases Menkes Disease and Wilsonâs Disease. The diseases are caused by mutations in the copper-transporting ATPases ATP7A and ATP7B, respectively. ATP7A and ATP7B are P-Type ATPases that both feature six N-Terminal metal binding domains (MBDs) that sense cytosolic copper concentrations through interactions with the copper chaperone ATOX1 and regulate ATPase activity and localization accordingly. The mechanism by which the N-Termini integrate and relay this information is poorly understood. The research presented here uses biochemical and computational techniques to characterize the organization and roles of the individual MBDs within the N-Terminal domains of the Cu-ATPases. We present evidence that the N-Terminal MBDs of ATP7B (N-ATP7B) are organized into a close-packed structure that allows for minor structural perturbations, such as copper binding to the CxxC motif of an MBD, to result in significant domain reorganization. This close packing can also be disrupted by the mutation of the CxxC motifs of MBDs 2,3,4 or 6 to AxxA. For MBD2 and MBD3, this mutation is also accompanied by an increased susceptibility of the domain to oxidation. However, when both MBD2 and 3 are mutated in tandem, the domain retains normal redox activity, suggesting the two MBDs work in concert. In contrast to the AxxA substitutions, when the CxxC of MBD2 is mutated to SxxS, N-ATP7B retains the conformation and reduction state of the wild-type protein, suggesting that hydrogen bonding plays a role in inter-MBD communication. MBD2 has been previously shown to be the primary site of copper transfer from ATOX1. Our data suggests that the organization of the MBDs in the Apo state favors transfer to MBD2, while MBD4 and MBD6 are likely to receive copper as downstream targets. The chaperone Atox1 transfers copper to N-ATP7A and N-ATP7B through the formation of heterodimers with individual MBDs. We show that ATOX1 is capable of transferring copper to all six MBDs of both N-Terminal domains, but that N-ATP7B can be fully metallated with a significantly lower concentration of Cu-ATOX1. ATOX1 may fully metallate N-ATP7B with repeat exposures at lower concentrations, and shows preferential target MBDs within both N-Termini. N-ATP7A shows reduced mobility upon copper transfer, but unlike N-ATP7B, does not show an altered proteolysis pattern with increased copper bound. These data suggest the two N-Terminal domains have different structural responses to copper binding, with N-ATP7B possessing a degree of cooperativity not seen for N-ATP7A. This cooperativity may be altered by the Wilsonâs disease causing mutants G85V and G591D, as they both show alterations in the concentration dependence of copper transfer from Cu-ATOX1. Cooperativity and reorganization are likely to be mediated by the inter-MBD loop regions. These loops contain sequences necessary for inter-domain and inter-protein interactions that regulate ATPase activity and trafficking. Our data suggests that copper transfer to N-ATP7B triggers a sequential set of conformational changes resulting in differential exposure of key loop residues. This mechanism provides the basis for copper dependent regulation of ATP7B.