Copper is an integral component of key metabolic enzymes. Numerous physiological processes depend on a fine balance between the biosynthetic incorporation of copper into proteins and the export of excess copper from the cell. The homeostatic control of copper requires the activity of the copper transporting ATPases (Cu-ATPases). In Wilson disease the disruption in the function of the Cu-ATPase ATP7B results in the accumulation of excess copper and a marked deficiency of copper-dependent enzymes. In this work, the structure of ATP7B has been modeled by homology using the Ca-ATPase X-ray structure, enabling a mechanism of copper transport by ATP7B to be proposed. The fourth transmembrane helix (TM4) of Ca-ATPase contains conserved residues critical to cation binding and is predicted to correspond to TM6 of the ATP7B homology model, containing the highly conserved CXXCPC motif. The interaction with Cu(I) and the importance of the 3 cysteines in TM6 of ATP7B has been shown using model peptides. ATP7B has a large cytoplasmic N-terminus comprised of six copper-binding domains (WCBD1-6), each capable of binding one Cu(I). Protein-protein interactions between WCBDs and the copper chaperone Atox1 has been shown, contrary to previous reports, to occur even in the absence of copper. 15N relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is minimal change in the dynamic properties and the relative orientation of the domains in the two states. The domain 4-5 linker remains flexible, and domain 5-6 is not a rigid dimer, with flexibility between the domains. Copper transfer to and between WCBD1-6 likely occurs via protein interactions facilitated by the flexibility of the domains with respect to each other. The flexible linkers connecting the domains are important in giving the domains motional freedom to interact with Atox1, to transfer copper to other domains, and finally to transfer copper to the transmembrane site for transport across the membrane.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/31746 |
| Date | 06 January 2012 |
| Creators | Fatemi, Negah |
| Contributors | Forman-Kay, Julie Deborah, Sarkar, Bibudhendra |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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