Cu(I) affinities of the domain 1 and 3 sites in the human metallochaperone for Cu,Zn-superoxide dismutase.
The delivery of copper by the human metallochaperone CCS is a key step in the activation of Cu,Zn-superoxide dismutase (SOD1). CCS is a three-domain protein with Cu(I)-binding CXXC and CXC motifs in domains 1 and 3, respectively. A detailed analysis of the binding of copper to CCS, including variants in which the Cys residues from domains 1 and 3 have been mutated to Ser, and also using separate domain 1 and 3 constructs, demonstrates that CCS is able to bind 1 equiv of Cu(I) in both of these domains. The Cu(I) affinity of domain 1 is approximately 5 × 10(17) M(-1) at pH 7.5, while that of domain 3 is at least 1 order of magnitude weaker. The CXXC site will therefore be preferentially loaded with Cu(I), suggesting that domain 1 plays a role in the acquisition of the metal. The delivery of copper to the target occurs via domain 3 whose structural flexibility and ability to be transiently metalated during copper delivery appear to be more important than the Cu(I) affinity of its CXC motif. The Cu(I) affinity of domain 1 of CCS is comparable to that of HAH1, another cytosolic copper metallochaperone. CCS and HAH1 readily exchange Cu(I), providing a mechanism whereby cross-talk can occur between copper trafficking pathways.
Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom.
Stephen
S
Allen
Adriana
A
Badarau
Christopher
C
Dennison
D020816:Amino Acid Motifs; D001665:Binding Sites; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013054:Spectrophotometry, Atomic / Q000379:methods; D013482:Superoxide Dismutase / Q000737:chemistry
D012694:Serine; D003300:Copper; D013482:Superoxide Dismutase; D003545:Cysteine