title: Copper trafficking mechanism of CXXC-containing domains: insight from the pH-dependence of their Cu(I) affinities.
abstract: Copper is trafficked to cellular destinations by homeostatic proteins that also prevent adverse reactivity of the metal. The copper metallochaperone HAH1 (human Atx1) binds Cu(I) via a CXXC motif on loop1/α1 of a βαββαβ ferredoxin-like structure. A similar fold constitutes each of the six metal-binding domains (MBDs) of the two P-type ATPases (Menkes and Wilson disease proteins), the destination for copper bound to HAH1. In this work we have investigated the influence of pH on copper trafficking between HAH1 and the first MBD of the Menkes protein (MNK1). Cu(I) affinities of 5.6 × 10(17) and 3.6 × 10(17) M(-1) have been determined at pH 7.0 for HAH1 and MNK1, respectively, from competition titrations with the chromophoric Cu(I) ligand bathocuproine disulfonate. The mutation of Lys60 on loop5 of HAH1 to Ala (the corresponding residue is Phe67 in MNK1) results in a 3-fold lowering of the affinity for Cu(I) at pH 7.0. The Cu(I) affinity of WT HAH1 exhibits a different pH-dependence compared to MNK1 and Lys60Ala HAH1. This arises because the pK(a) of the second Cys ligand in the CXXC motif of HAH1 is 1.5 pH units lower due to stabilization of the thiolate via a hydrogen-bonding interaction with the side chain of Lys60. The thermodynamic gradient for Cu(I) transfer between HAH1 and MNK1 depends on pH. The decrease in the pK(a) of the Cys ligand in HAH1 can also influence the kinetics of Cu(I) transfer.
authors.affiliation: Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Adriana
authors.initials: A
authors.lastname: Badarau
authors.forename: Christopher
authors.initials: C
authors.lastname: Dennison
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001419:Bacteria / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D027682:Cation Transport Proteins / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000079922:Copper Transport Proteins; D000073840:Copper-Transporting ATPases; D005656:Fungal Proteins / Q000378:metabolism; D005658:Fungi / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D056511:Metallochaperones; D018832:Molecular Chaperones / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: C474500:ATOX1 protein, human; D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D005656:Fungal Proteins; D056511:Metallochaperones; D018832:Molecular Chaperones; D003300:Copper; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases