A Multinuclear Copper(I) Cluster Forms the Dimerization Interface in Copper-Loaded Human Copper Chaperone for Superoxide Dismutase
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.
- Research Organization:
- SLAC National Accelerator Lab., Menlo Park, CA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC02-76SF00515
- OSTI ID:
- 953891
- Report Number(s):
- SLAC-REPRINT-2009-501; TRN: US201004%%628
- Journal Information:
- Biochem.46:11845,2007, Vol. 6
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
ABSORPTION SPECTROSCOPY
ATOMIC CLUSTERS
BIOCHEMISTRY
COORDINATES
COPPER
DIMERIZATION
DIMERS
INTERACTIONS
INTERFACES
LOADING
MALTOSE
METALS
MUTANTS
MUTATIONS
POLYPEPTIDES
PROTEINS
REACTIVITY
SPECTROSCOPY
SUPEROXIDE DISMUTASE
VECTORS
Other
BIO
CHEM