title: Stoichiometry of complex formation between Copper(I) and the N-terminal domain of the Menkes protein.
abstract: The inherent cellular toxicity of copper ions demands that their concentration be carefully controlled. The cellular location of the Menkes ATPase, a key element in the control of intracellular copper, is regulated by the intracellular copper concentration through the N-terminus of the enzyme, comprising 6 homologous subdomains or modules, each approximately 70 residues in length and containing a -Cys-X-X-Cys- motif. Based on the proposal that binding of copper to these modules regulates the Menkes ATPase cellular location by promoting changes in the tertiary structure of the enzyme, we have expressed the entire N-terminal domain (MNKr) and the second metal-binding module (MNKr2) of the Menkes protein in E. coli and purified them to homogeneity. Ultraviolet-visible, luminescence, and X-ray absorption spectroscopy show that copper and silver bind to the single module, MNKr2, with a stoichiometry of one metal ion per module. However, the array of six modules, MNKr, binds Cu(I) to produce a homogeneous conformer with 4 mol equiv of metal ion. The metal ions are bound in an environment that is shielded from solvent molecules. We suggest a model of the Menkes protein in which the Cu(I) binding induces tertiary changes in the organization of the six metal-binding domains.
authors.affiliation: National Research Center for Environmental Toxicology, University of Queensland, Coopers Plains, Queensland 4108, Australia.
authors.forename: P A
authors.initials: PA
authors.lastname: Cobine
authors.forename: G N
authors.initials: GN
authors.lastname: George
authors.forename: D J
authors.initials: DJ
authors.lastname: Winzor
authors.forename: M D
authors.initials: MD
authors.lastname: Harrison
authors.forename: S
authors.initials: S
authors.lastname: Mogahaddas
authors.forename: C T
authors.initials: CT
authors.lastname: Dameron
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D027682:Cation Transport Proteins; D003300:Copper / Q000737:chemistry*; D000073840:Copper-Transporting ATPases; D005583:Fourier Analysis; D006801:Humans; D008163:Luminescent Measurements; D007706:Menkes Kinky Hair Syndrome / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000737:chemistry; D012834:Silver / Q000737:chemistry; D013053:Spectrophotometry; D014461:Ultracentrifugation
chemical_list: D002352:Carrier Proteins; D027682:Cation Transport Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012834:Silver; D003300:Copper; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases