Copper transfer studies between the N-terminal copper binding domains one and four of human Wilson protein.
Human Wilson protein functions in the secretory pathway to insert copper ultimately into the multicopper oxidase ceruloplasmin and also plays a role in the excretion of excess copper to the bile. This copper-transporting P-type ATPase possesses six N-terminal cytosolic copper-binding domains contained within an approximately 72 amino acid consensus motif and the first four of these domains, denoted WLN1-4, are implicated in copper acquisition from the metallochaperone HAH1, whereas the domains closest to the membrane portion of the enzyme, WLN5-6, are essential for copper transport across the membrane. In order to test our hypothesis that copper transfer occurs between domains in the N-terminus of Wilson protein, we expressed and purified to homogeneity copper-binding domains 1, 3, 4, 5-6, and 6, denoted by WLN1, WLN3, WLN4, WLN5-6, and WLN6, respectively. Since we determined WLN1 and WLN4 to have the highest and lowest isoelectric points (6.77 and 3.85, respectively) and thus are readily separated via ion exchange chromatography, we developed a copper transfer assay between these domains. We anaerobically incubated either Cu(I)-WLN1 with apo-WLN4 or apo-WLN1 with Cu(I)-WLN4, then separated these domains and quantified the amount of copper that migrates from one domain to another by ICP-MS. Regardless of whether we start with Cu(I)-WLN1 or Cu(I)-WLN4 as the initial copper donor, we demonstrate facile copper transfer between WLN1 and WLN4, thereby demonstrating the feasibility of copper transfer between these domains in vivo.
Bunce
Jennifer
J
Department of Chemistry, Western Michigan University, 1903 W. Michigan Ave., Kalamazoo, MI 49008-5413, USA.
Achila
David
D
Hetrick
Evan
E
Lesley
Leighann
L
Huffman
David L
DL
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D006801:Humans; D007526:Isoelectric Point; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D027682:Cation Transport Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases