Zinc-mediated interaction of copper chaperones through their heavy-metal associated domains

Background: A copper chaperone CCS is a multi-domain protein that supplies a copper ion to Cu/Zn-superoxide dismutase (SOD1). Among the domains of CCS, the N-terminal domain (CCS^dI) belongs to a heavy metal-associated (HMA) domain, in which a Cys-x-x-Cys (CxxC) motif binds a heavy metal ion. It has hence been expected that the HMA domain in CCS has a role in the metal trafficking; however, the CxxC motif in the domain is dispensable for supplying a copper ion to SOD1, leaving an open question on roles of CCS^dI in CCS. Methods: To evaluate protein-protein interactions of CCS through CCS^dI, yeast two-hybrid assay, a pull-down assay using recombinant proteins, and the analysis with fluorescence resonance energy transfer were performed. Results: We found that CCS specifically interacted with another copper chaperone HAH1, a HMA domain protein, through CCS^dI. The interaction between CCS^dI and HAH1 was not involved in the copper supply from CCS to SOD1 but was mediated by a zinc ion ligated with Cys residues of the CxxC motifs in CCS^dI and HAH1. Conclusion: While physiological significance of the interaction between copper chaperones awaits further investigation, we propose that CCS^dI would have a role in the metal-mediated interaction with other proteins including heterologous copper chaperones.