Mechanism of Cu + -transporting ATPases: Soluble Cu + chaperones directly transfer Cu + to transmembrane transport sites

Abstract

As in other P-type ATPases, metal binding to transmembrane metal-binding sites (TM-MBS) in Cu + -ATPases is required for enzyme phosphorylation and subsequent transport. However, Cu + does not access Cu + -ATPases in a free (hydrated) form but is bound to a chaperone protein. Cu + transfer from Cu + chaperones to regulatory cytoplasmic metal-binding domains (MBDs) present in these ATPases has been described, but there is no evidence of a proposed subsequent Cu + movement from the MBDs to the TM-MBS. Alternatively, we postulate the parsimonious Cu + transfer by the chaperone directly to TM-MBS. Testing both models, the delivery of Cu + by Archaeoglobus fulgidus Cu + chaperone CopZ to the corresponding Cu + -ATPase, CopA, was studied. As expected, CopZ interacted with and delivered the metal to CopA MBDs. Cu + -loaded MBDs, acting as metal donors, were unable to activate CopA or a truncated CopA lacking MBDs. Conversely, Cu + -loaded CopZ activated the CopA ATPase and CopA constructs in which MBDs were rendered unable to bind Cu + . Furthermore, under nonturnover conditions, CopZ transferred Cu + to the TM-MBS of a CopA lacking MBDs. These data are consistent with a model where MBDs serve a regulatory function without participating in metal transport and the chaperone delivers Cu + directly to transmembrane transport sites of Cu + -ATPases.