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.

FIGURE 1.

Scheme of CopA structure (A) and transmembrane Cu⁺-binding sites (B). The topological scheme of CopA includes the A-domain (PDB 2HC8) and ATP-BD (PDB 2B8E) structures. N-MBD and C-MBD structures are homology models based on the fourth N-MBD of the Menkes protein (PDB 1AW0) and in Bacillus subtilis CopZ (PDB 1K0V), respectively. Constructs ΔC-CopA and ΔN,C-CopA lack one or both MBD domains. C₂-CopA has Ala substitutions in the Cys present in N-MBD and C-MBD. C₂^N-MBD-CopA has Ala substitutions in the Cys present in TM-MBSs and C-MBD. C₀-CopA has all Cys in the molecule replaced by Ala. Amino acids involved in the Cu⁺ coordination by the TM-MBS are indicated in B.

FIGURE 2.

Interaction between N-MBD and ATP-BD. A, SDS-PAGE of a representative co-purification assay between ATP-BD and N-MBD or Cu⁺-loaded N-MBD. B, SDS-PAGE of a representative co-purification assay between N-MBD and ATP-BD in the absence and in the presence of 5 mm ADP-Mg²⁺. 40% of unbound protein (UP) or 40% of the bound protein (BP) fractions were loaded in each lane.

FIGURE 3.

Interaction between N-MBD and A-domain. SDS-PAGE of a representative co-purification assay between A-domain and N-MBD or Cu⁺-loaded N-MBD. 40% of unbound protein (UP) or 40% of the bound protein (BP) fractions were loaded in each lane.

FIGURE 4.

Proposed catalytic and transport cycle of Cu⁺-ATPases.