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

Abstract

BackgroundA 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 (CCSdI) 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 CCSdI in CCS. MethodsTo evaluate protein-protein interactions of CCS through CCSdI, yeast two-hybrid assay, a pull-down assay using recombinant proteins, and the analysis with fluorescence resonance energy transfer were performed. ResultsWe found that CCS specifically interacted with another copper chaperone HAH1, a HMA domain protein, through CCSdI. The interaction between CCSdI 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 CCSdI and HAH1. ConclusionWhile physiological significance of the interaction between copper chaperones awaits further investigation, we propose that CCSdI would have a role in the metal-mediated interaction with other proteins including heterologous copper chaperones.