Identification of a copper-induced intramolecular interaction in the transcription factor Mac1 from Saccharomyces cerevisiae

Mad mediates copper (Cu)-dependent expression of genes involved in high-affinity uptake of copper ions in Saccharomyces cerevisiae. Mad is a transcriptional activator in Cu-deficient cells, but is inhibited in Cu-replete cells. Mad resides within the nucleus in both Cu-deficient and Cu-loaded cells. Cu inhibition of Mad appears to result from binding of eight copper ions within a C-terminal segment consisting of two Cys-rich motifs. In addition, two zinc ions are bound within the N-terminal DNA-binding domain. Only 4-5 mel. eq. Cu are bound to a mutant Mad (His279Gln substitution) that is impervious to Cu inhibition. The CuMac1 complex is luminescent, indicative of copper bound in the Cu(I) state. Cu binding induces a molecular switch resulting in an intramolecular interaction in Mad between the N-terminal DNA-binding domain and the C-terminal activation domain. This allosteric interaction is Cu dependent and is not observed when Mad contained the mutant His279Gln substitution. Fusion of the minimal DNA-binding domain of Mac1 (residues 1-159) to the minimal Cu-binding activation domain (residues 252-341) yields a functional Cu-regulated transcriptional activator. These results suggest that Cu repression of Mac1 arises from a Cu-induced intramolecular interaction that inhibits both DNA binding and transactivation activities.