Conserved residues modulate copper release in human copper chaperone Atox1.
It is unclear how the human copper (Cu) chaperone Atox1 delivers Cu to metal-binding domains of Wilson and Menkes disease proteins in the cytoplasm. To begin to address this problem, we have characterized Cu(I) release from wild-type Atox1 and two point mutants (Met(10)Ala and Lys(60)Ala). The dynamics of Cu(I) displacement from holo-Atox1 were measured by using the Cu(I) chelator bicinchonic acid (BCA) as a metal acceptor. BCA removes Cu(I) from Atox1 in a three-step process involving the bimolecular formation of an initial Atox1-Cu-BCA complex followed by dissociation of Atox1 and the binding of a second BCA to generate apo-Atox1 and Cu-BCA(2). Both mutants lose Cu(I) more readily than wild-type Atox1 because of more rapid and facile displacement of the protein from the Atox1-Cu-BCA intermediate by the second BCA. Remarkably, Cu(I) uptake from solution by BCA is much slower than the transfer from holo-Atox1, presumably because of slow dissociation of DTT-Cu complexes. These results suggest that Cu chaperones play a key role in making Cu(I) rapidly accessible to substrates and that the activated protein-metal-chelator complex may kinetically mimic the ternary chaperone-metal-target complex involved in Cu(I) transfer in vivo.
Hussain
Faiza
F
Department of Biochemistry and Cell Biology, Keck Center for Structural Computational Biology, Houston, TX 77251, USA.
Olson
John S
JS
Wittung-Stafshede
Pernilla
P
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D000079922:Copper Transport Proteins; D000073840:Copper-Transporting ATPases; D006801:Humans; D007700:Kinetics; D056511:Metallochaperones; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000502:physiology; D011804:Quinolines / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C474500:ATOX1 protein, human; D027682:Cation Transport Proteins; D002614:Chelating Agents; D000079922:Copper Transport Proteins; D056511:Metallochaperones; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; D011804:Quinolines; D012710:Serum Albumin, Bovine; D003300:Copper; C047117:bicinchoninic acid; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases