Identification of a copper-induced intramolecular interaction in the transcription factor Mac1 from Saccharomyces cerevisiae.
Mac1 mediates copper (Cu)-dependent expression of genes involved in high-affinity uptake of copper ions in Saccharomyces cerevisiae. Mac1 is a transcriptional activator in Cu-deficient cells, but is inhibited in Cu-replete cells. Mac1 resides within the nucleus in both Cu-deficient and Cu-loaded cells. Cu inhibition of Mac1 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 mol. eq. Cu are bound to a mutant Mac1 (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 Mac1 between the N-terminal DNA-binding domain and the C-terminal activation domain. This allosteric interaction is Cu dependent and is not observed when Mac1 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.
Jensen
L T
LT
Departments of Biochemistry and Medicine, University of Utah Health Science Center, Salt Lake City, UT 84132, USA.
Winge
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DR
D019943:Amino Acid Substitution; D003300:Copper / Q000378:metabolism / Q000494:pharmacology*; D003545:Cysteine; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D008164:Luminescent Proteins; C084954:MAC1 protein, S cerevisiae; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D003300:Copper; D003545:Cysteine