NMR characterization of copper-binding domains 4-6 of ATP7B .
The Wilson disease protein (ATP7B) is a copper-transporting member of the P-type ATPase superfamily, which plays a central role in copper homeostasis and interacts with the copper chaperone Atox1. The N-terminus of ATP7B is comprised of six copper-binding domains (WCBDs), each capable of binding one copper atom in the +1 oxidation state. To better understand the regulatory effect of copper binding to these domains, we have performed NMR characterization of WCBD4-6 (domains 4-6 of ATP7B). (15)N relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is no dramatic change in the dynamic properties of this three-domain construct; the linker between domains 4 and 5 remains flexible, domains 5 and 6 do not form a completely rigid dimer but rather have some flexibility with respect to each other, and there is minimal change in the relative orientation of the domains in the two states. We also show that, contrary to previous reports, the protein-protein interaction between Atox1 and the copper-binding domains takes place even in the absence of copper. Comparison of apo and Cu(I)-bound spectra of WCBD1-6 shows that binding of Cu(I) does not induce the formation of a unit that tumbles as a single entity, consistent with our results for WCBD4-6. We propose that copper transfer to and between the N-terminal domains of the Wilson Cu-ATPase occurs via protein interactions that are facilitated by the flexibility of the linkers and the motional freedom of the domains with respect to each other.
Fatemi
Negah
N
Department of Biochemistry, University of Toronto, Toronto,Ontario M5S 1A8, Canada.
Korzhnev
Dmitry M
DM
Velyvis
Algirdas
A
Sarkar
Bibudhendra
B
Forman-Kay
Julie D
JD
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000079922:Copper Transport Proteins; D000073840:Copper-Transporting ATPases; D006801:Humans; D056511:Metallochaperones; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary
C474500:ATOX1 protein, human; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D056511:Metallochaperones; D018832:Molecular Chaperones; D003300:Copper; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases