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  • Title: Zn(II) binding and DNA binding properties of ligand-substituted CXHH-type zinc finger proteins.
    Author: Imanishi M, Matsumura K, Tsuji S, Nakaya T, Negi S, Futaki S, Sugiura Y.
    Journal: Biochemistry; 2012 Apr 24; 51(16):3342-8. PubMed ID: 22482427.
    Abstract:
    CCHH-type zinc fingers are among the most common DNA binding motifs found in eukaryotes. In a previous report, we substituted the second ligand cysteine residue with aspartic acid, producing a Zn(II)-responsive transcription factor; this indicates that a ligand substitution is a possible design target of an engineered zinc finger peptide. Despite the importance of Zn(II) binding with respect to the folding and DNA binding properties of a zinc finger peptide, no study about the effects of ligand substitution on both Zn(II) binding and DNA binding properties has been reported. Here, we substituted a conserved cysteine (C) with other zinc-coordinated amino acid residues, histidine (H), aspartic acid (D), and glutamic acid (E), to create CXHH-type zinc finger peptides (X = C, H, D, and E). The Zn(II)-dependent conformational change was observed in all peptides; however, the Zn(II) binding affinity and metal coordination geometry of the peptides were different. Gel mobility shift assays showed that the Zn(II)-bound forms of the ligand-substituted derivatives retain DNA binding ability, while the DNA binding affinity decreased in the following manner: CCHH > CDHH > CEHH ≫ CHHH. The DNA binding sequence preferences of the ligand-substituted derivatives were similar to that of the wild type in the context of the full three-finger DNA-binding domain of transcription factor Zif268. These results indicate that artificial zinc finger proteins with various DNA binding affinities that respond to a diverse range of Zn(II) concentrations can be designed by substituting the Zn(II) ligand.
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