Cu(I) affinities of the domain 1 and 3 sites in the human metallochaperone for Cu,Zn-superoxide dismutase

Biochemistry. 2012 Feb 21;51(7):1439-48. doi: 10.1021/bi201370r. Epub 2012 Feb 9.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Binding Sites
  • Chromatography, Gel
  • Cloning, Molecular
  • Copper / chemistry*
  • Cysteine / chemistry
  • Cytosol / chemistry
  • Dose-Response Relationship, Drug
  • Escherichia coli / metabolism*
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Structure, Tertiary
  • Serine / chemistry
  • Spectrophotometry, Atomic / methods
  • Superoxide Dismutase / chemistry

Substances

  • Serine
  • Copper
  • Superoxide Dismutase
  • Cysteine