The DNA binding domain of GAL4 forms a binuclear metal ion complex.
The transcription factor GAL4 from Saccharomyces cerevisiae requires Zn(II) or Cd(II) for specific recognition of the UASG sequence (Pan & Coleman, 1989). An N-terminal fragment consisting of the first 63 amino acid residues of GAL4 [GAL4(63)] has been obtained by partial tryptic proteolysis of a cloned and overproduced N-terminal domain of 149 residues, GAL(149). We show that GAL4(63) contains the minimal GAL4 DNA binding domain. GAL4(63) binds tightly 1-2 mol of Zn(II) or 2 mol of Cd(II). 113Cd NMR of 113Cd(II)-substituted GAL4(63) reveals structural identity between the metal binding domains of GAL4(63) and that of the larger precursor GAL4(149). 113Cd(II) can be substituted for the Zn(II) in GAL4(63), and two 113Cd NMR signals are observed at 706 and 669 ppm, both suggesting coordination of 113Cd(II) to three or four -S- ligands. With the exception of the N-terminal methionine, the only sulfur-containing residues are the six highly conserved cysteines. High-resolution 1H NMR of Zn(II)-GAL4(63) and Cd(II)-GAL4(63) show the two proteins to have almost identical conformations and to be present as monomers in solutions up to millimolar concentration. This leads us to postulate that GAL4 does not possess a TFIIIA-like "Zn-finger" but forms a binuclear metal cluster involving all six cysteines in a "cloverleaf"-like array. GAL4(63) contains about 60% alpha-helix, estimated from circular dichroism. Removal of the native Zn(II) causes substantial unfolding of the secondary structure. Unlike GAL4(149), the resultant apoprotein is not induced to refold by readdition of Zn(II) at low concentrations.
Pan
T
T
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
Coleman
J E
JE
D000595:Amino Acid Sequence; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D015032:Zinc / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D010446:Peptide Fragments; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D002104:Cadmium; D004247:DNA; D015032:Zinc