Sequential assignments of the 1H NMR resonances of Zn(II)2 and 113Cd(II)2 derivatives of the DNA-binding domain of the GAL4 transcription factor reveal a novel structural motif for specific DNA recognition.
The DNA-binding domain of the GAL4 transcription factor, consisting of the 62 N-terminal amino acid residues and denoted GAL4(62*), contains a novel Zn(II)2Cys6 or Cd(II)2Cys6 binuclear cluster [Pan, T., & Coleman, J. E. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2077]. Specific DNA recognition requires residues located within as well as C terminal to this binuclear cluster. 1H NMR sequential assignments have been carried out on Zn(II)2- and 113Cd(II)2GAL4(62*) by using DQF-COSY, relayed COSY, double-relayed COSY, and NOESY. The ligands of the two tetrahedral metal-binding sites have been identified as Cys11, Cys14, Cys21, and Cys31 to one metal ion and Cys28, Cys38, Cys21, and Cys31 to the other metal ion with Cys21 and Cys31 as ligands shared between the two metal ions. No alpha-helices can be found within the GAL4(62*) structure, which consists of a series of turns to accommodate the metal cluster, followed by irregular loops and turns from residues 42 to 60, the "specificity region", whose sequence contributes importantly to specific DNA recognition. Long-distance NOE's are observed between residues forming the binuclear cluster and several residues within the specificity region, indicating that the latter is folded compactly onto the metal cluster. The requirement of the Zn(II)2Cys6 binuclear cluster and the specificity region for binding to DNA reveals GAL4 as a member of a class of specific DNA-binding proteins using a new structural motif for the recognition of specific DNA sequences. Specific DNA binding by this class of proteins is achieved by use of turns and loops that enclose a Zn(II)2Cys6 binuclear cluster, instead of alpha-helices or beta-strands as observed in specific DNA-binding proteins described previously.
Pan
T
T
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Coleman
J E
JE
D000595:Amino Acid Sequence; D002104:Cadmium / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D002104:Cadmium; D004247:DNA; D015032:Zinc