Multiple protein domains contribute to the action of the copper chaperone for superoxide dismutase

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 copp er chaperone for SOD1 (CCS): an amino terminal Domain I homologous to the A tx1p metallochaperone, a central portion (Domain II) homologous to SOD1, an d a short carboxyl-terminal peptide unique to CCS molecules (Domain III), T hese regions fold into distinct polypeptide domains as revealed through pro teolysis protection studies. The biological roles of the yeast CCS domains were examined in yeast cells. Surprisingly, Domain I was found to be necess ary only under conditions of strict copper limitation. Domain I and Atx1p w ere not interchangeable in vivo, underscoring the specificity of the corres ponding metallochaperones. A putative copper site in Domain II was found to be irrelevant to yeast CCS activity, but SOD1 activation invariably requir ed a CXC in Domain III that binds copper. Copper binding to purified yeast CCS induced allosteric conformational changes in Domain III and also enhanc ed homodimer formation of the polypeptide, Our results are consistent with a model whereby Domain I recruits cellular copper, Domain II facilitates ta rget recognition, and Domain III, perhaps in concert with Domain I, mediate s copper insertion into apo-SOD1.