A multinuclear copper(I) cluster forms the dimerization interface in copper-loaded human copper chaperone for superoxide dismutase

Biochemistry. 2007 Oct 23;46(42):11845-56. doi: 10.1021/bi700566h. Epub 2007 Sep 29.


Copper binding and X-ray aborption spectroscopy studies are reported on untagged human CCS (hCCS; CCS = copper chaperone for superoxide dismutase) isolated using an intein self-cleaving vector and on single and double Cys to Ala mutants of the hCCS MTCQSC and CSC motifs of domains 1 (D1) and 3 (D3), respectively. The results on the wild-type protein confirmed earlier findings on the CCS-MBP (maltose binding protein) constructs, namely, that Cu(I) coordinates to the CXC motif, forming a cluster at the interface of two D3 polypeptides. In contrast to the single Cys to Ser mutations of the CCS-MBP protein (Stasser, J. P., Eisses, J. F., Barry, A. N., Kaplan, J. H., and Blackburn, N. J. (2005) Biochemistry 44, 3143-3152), single Cys to Ala mutations in D3 were sufficient to eliminate cluster formation and significantly reduce CCS activity. Analysis of the intensity of the Cu-Cu cluster interaction in C244A, C246A, and C244/246A variants suggested that the nuclearity of the cluster was greater than 2 and was most consistent with a Cu4S6 adamantane-type species. The relationship among cluster formation, oligomerization, and metal loading was evaluated. The results support a model in which Cu(I) binding converts the apo dimer with a D2-D2 interface to a new dimer connected by cluster formation at two D3 CSC motifs. The predominance of dimer over tetramer in the cluster-containing species strongly suggests that the D2 dimer interface remains open and available for sequestering an SOD1 monomer. This work implicates the copper cluster in the reactive form and adds detail to the cluster nuclearity and how copper loading affects the oligomerization states and reactivity of CCS for its partner SOD1.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Absorptiometry, Photon
  • Alanine / metabolism
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Chromatography, High Pressure Liquid
  • Copper / chemistry
  • Copper / metabolism*
  • Dimerization
  • Escherichia coli / genetics
  • Fourier Analysis
  • Genetic Variation
  • Humans
  • Models, Molecular
  • Molecular Chaperones / chemistry
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism*
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / metabolism
  • Sequence Analysis, DNA
  • Superoxide Dismutase / genetics
  • Superoxide Dismutase / metabolism*
  • Zinc / chemistry
  • Zinc / metabolism


  • Molecular Chaperones
  • Recombinant Fusion Proteins
  • Copper
  • Superoxide Dismutase
  • Zinc
  • Alanine