title: Zn(II) binding and DNA binding properties of ligand-substituted CXHH-type zinc finger proteins.
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.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. imiki@scl.kyoto-u.ac.jp
authors.forename: Miki
authors.initials: M
authors.lastname: Imanishi
authors.forename: Kazushi
authors.initials: K
authors.lastname: Matsumura
authors.forename: Shogo
authors.initials: S
authors.lastname: Tsuji
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Nakaya
authors.forename: Shigeru
authors.initials: S
authors.lastname: Negi
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
authors.forename: Yukio
authors.initials: Y
authors.lastname: Sugiura
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D007700:Kinetics; D008024:Ligands; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*; D016335:Zinc Fingers
chemical_list: D008024:Ligands; D010455:Peptides; D004247:DNA; D015032:Zinc; D003545:Cysteine