title: Chaperone-mediated Cu+ delivery to Cu+ transport ATPases: requirement of nucleotide binding.
abstract: Cu(+)-ATPases drive the efflux of Cu(+) from the cell cytoplasm. During their catalytic/transport cycle, cytoplasmic Cu(+)-chaperones deliver the metal to the two transmembrane metal-binding sites (TM-MBSs) responsible for Cu(+) translocation. Here, using Archaeoglobus fulgidus Cu(+)-ATPase CopA and the C-terminal Cu(+)-chaperone domain of CopZ (Ct-CopZ), we describe the mechanism of Cu(+) transfer to both TM-MBSs. In absence of other ligands, Ct-CopZ transfers Cu(+) to wild-type CopA and to various CopA constructs lacking or having mutated cytoplasmic metal-binding domains, in a fashion consistent with occupancy of a single TM-MBS. Similar experiments performed in the presence of 2.5 mm ADP-Mg(2+), stabilizing an E1.ADP, lead to full occupancy of both TM-MBSs. In both cases, the transfer is largely stoichiometric, i.e. equimolar amounts of Ct-CopZ.Cu(+) saturated the TM-MBSs. Experiments performed with CopA mutants lacking either TM-MBS showed that both sites are loaded independently, and nucleotide binding does not affect their availability. The nucleotide-induced E2-->E1 transition is structurally characterized by a large displacement of the A and N domains opening the cytoplasmic region of P-type ATPases. Then, it is apparent that, whereas the first Cu(+)-chaperone can bind an ATPase form available in the absence of ligands, the second requires the E1.nucleotide intermediary to interact and deliver the metal. Interestingly, independent of TM-MBS Cu(+) loading, nucleotide binding also prevents the regulatory interaction of the N-terminal cytoplasmic metal-binding domain with the nucleotide binding domain.
authors.affiliation: Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA.
authors.forename: Manuel
authors.initials: M
authors.lastname: González-Guerrero
authors.forename: Deli
authors.initials: D
authors.lastname: Hong
authors.forename: José M
authors.initials: JM
authors.lastname: Argüello
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D001665:Binding Sites; D055162:Biocatalysis; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D027682:Cation Transport Proteins; D018832:Molecular Chaperones; D009711:Nucleotides; D003300:Copper; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases