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Zn(II) Binding and DNA Binding Properties of Ligand-Substituted CXHH-Type Zinc Finger Proteins

Author:
Imanishi, Miki, Matsumura, Kazushi, Tsuji, Shogo, Nakaya, Tomohiro, Negi, Shigeru, Futaki, Shiroh, Sugiura, Yukio
Source:
Biochemistry 2012 v.51 no.16 pp. 3342-3348
ISSN:
1520-4995
Subject:
DNA, aspartic acid, binding capacity, cysteine, eukaryotic cells, geometry, glutamic acid, histidine, peptides, transcription factors, zinc, zinc finger motif
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.
Agid:
5309568