Copper stabilizes a heterodimer of the yCCS metallochaperone and its target superoxide dismutase.
The copper chaperone for superoxide dismutase (CCS) activates the antioxidant enzyme Cu,Zn-SOD (SOD1) by directly inserting the copper cofactor into the apo form of SOD1. Neither the mechanism of protein-protein recognition nor of metal transfer is clear. The metal transfer step has been proposed to occur within a transient copper donor/acceptor complex that is either a heterodimer or heterotetramer (i.e. a dimer of dimers). To determine the nature of this intermediate, we generated a mutant form of SOD1 by replacing a copper binding residue His-48 with phenylalanine. This protein cannot accept copper from CCS but does form a stable complex with apo- and Cu-CCS, as observed by immunoprecipitation and native gel electrophoresis. Fluorescence anisotropy measurements corroborate the formation of this species and further indicate that copper enhances the stability of the dimer by an order of magnitude. The copper form of the heterodimer was isolated by gel filtration chromatography and contains one copper and one zinc atom per heterodimer. These results support a mechanism for copper transfer in which CCS and SOD1 dock via their highly conserved dimer interfaces in a manner that precisely orients the Cys-rich copper donor sites of CCS and the His-rich acceptor sites of SOD1 to form a copper-bridged intermediate.
Torres
A S
AS
Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, USA.
Petri
V
V
Rae
T D
TD
O'Halloran
T V
TV
D016880:Anisotropy; D001665:Binding Sites; D015153:Blotting, Western; D002850:Chromatography, Gel; D003300:Copper / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation; D010649:Phenylalanine / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
C491960:CCS1 protein, S cerevisiae; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; D010649:Phenylalanine; D006639:Histidine; D003300:Copper; D013482:Superoxide Dismutase