The in vitro analysis of metal binding and transfer by the copper metallochaperone for Cu/Zn-superoxide dismutase

Abstract

The human copper metallochaperone CCS activates Cu,Zn-superoxide dismutase (SOD1) through copper transfer and formation of an intramolecular disulfide. CCS is a three domain protein with CXXC and CXC Cu(I)-binding motifs in domains 1 and 3, respectively. A detailed analysis of copper binding to CCS and variants in which the Cys residues of the CXXC and CXC motifs in domains 1 and 3, respectively, have been mutated to Ser, and also domain 1 and 3 constructs, using the chromophoric Cu(I) ligands bathocuproine disulfonate and bicinchoninic acid has been performed. Competition experiments demonstrate that CCS is able to bind a single equivalent of Cu(I) independently in both domains 1 and 3. The Cu(I) affinity of domain 1 is approximately 5  1017 M-1 at pH 7.5 while that of domain 3 is at least an order of magnitude weaker. The order of magnitude difference between the Cu(I) affinities of domains 1 and 3, is maintained over the pH range ~6 to 9 and therefore, at physiological pH, in the cell cytosol, where copper is limiting, the CXXC site will be preferentially occupied with Cu(I). Under such conditions the transfer of Cu(I) from domain 1 to the buried active site of SOD1 must be facilitated by the structurally flexible domain 3. The mutation of Arg71, located on loop 5 of domain 1, to Ala results in an altered pH dependence of the Cu(I) affinity for the CXXC motif due to an increase in the pKa of the Cu(I)-binding C-terminal Cys (CysC) residue. Therefore, lowering of the CysC pKa through a hydrogen bond with the Arg71 side chain that stabilises the CysC thiolate in domain 1, observed in the crystal structure of Cu(I)-bound domain 1, modulates the nucleophilicity, and hence Cu(I)-binding affinity of this residue. In addition, high and low pKa’s for the domain 3 CXC motif Cys residues were also determined from the pH dependence of the Cu(I) affinity for this site. The relative nucleophilicities of the domain 1 and domain 3 Cu(I)-binding Cys residues, inferred from their pKa’s, suggest copper transfer from domain 1 to domain 3 is kinetically favoured. In addition, Zn(II) binding to the largest central domain of CCS was investigated to determine its role in affecting the quaternary structure of the protein. Zn(II)–binding to domain 2 was found to stabilise the dimeric form of the protein and also the CCS:SOD1 heterocomplex which orientates the Cu(I)-binding domains 1 and 3 of CCS for activation of SOD1.