title: Multiple protein domains contribute to the action of the copper chaperone for superoxide dismutase.
abstract: The copper chaperone for superoxide dismutase (SOD1) inserts the catalytic metal cofactor into SOD1 by an unknown mechanism. We demonstrate here that this process involves the cooperation of three distinct regions of the copper chaperone for SOD1 (CCS): an amino-terminal Domain I homologous to the Atx1p metallochaperone, a central portion (Domain II) homologous to SOD1, and a short carboxyl-terminal peptide unique to CCS molecules (Domain III). These regions fold into distinct polypeptide domains as revealed through proteolysis protection studies. The biological roles of the yeast CCS domains were examined in yeast cells. Surprisingly, Domain I was found to be necessary only under conditions of strict copper limitation. Domain I and Atx1p were not interchangeable in vivo, underscoring the specificity of the corresponding metallochaperones. A putative copper site in Domain II was found to be irrelevant to yeast CCS activity, but SOD1 activation invariably required a CXC in Domain III that binds copper. Copper binding to purified yeast CCS induced allosteric conformational changes in Domain III and also enhanced homodimer formation of the polypeptide. Our results are consistent with a model whereby Domain I recruits cellular copper, Domain II facilitates target recognition, and Domain III, perhaps in concert with Domain I, mediates copper insertion into apo-SOD1.
authors.affiliation: Departments of Environmental Health Sciences and Biochemistry, The Johns Hopkins University School of Public Health, Baltimore, Maryland 21205, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Schmidt
authors.forename: T D
authors.initials: TD
authors.lastname: Rae
authors.forename: R A
authors.initials: RA
authors.lastname: Pufahl
authors.forename: T
authors.initials: T
authors.lastname: Hamma
authors.forename: J
authors.initials: J
authors.lastname: Strain
authors.forename: T V
authors.initials: TV
authors.lastname: O'Halloran
authors.forename: V C
authors.initials: VC
authors.lastname: Culotta
mesh_terms: D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry* / Q000502:physiology; D019281:Dimerization; D018832:Molecular Chaperones / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013482:Superoxide Dismutase / Q000737:chemistry*
chemical_list: D018832:Molecular Chaperones; D003300:Copper; D013482:Superoxide Dismutase