title: An arginine residue instead of a conserved leucine residue in the recognition helix of the finger 3 of Zif268 stabilizes the domain structure and mediates DNA binding.
abstract: The Cys(2)His(2)-type zinc finger is a common DNA binding motif that is widely used in the design of artificial zinc finger proteins. In almost all Cys(2)His(2)-type zinc fingers, position 4 of the α-helical DNA-recognition site is occupied by a Leu residue involved in formation of the minimal hydrophobic core. However, the third zinc finger domain of native Zif268 contains an Arg residue instead of the conserved Leu. Our aim in the present study was to clarify the role of this Arg in the formation of a stable domain structure and in DNA binding by substituting it with a Lys, Leu, or Hgn, which have different terminal side-chain structures. Assessed were the metal binding properties, peptide conformations, and DNA-binding abilities of the mutants. All three mutant finger 3 peptides exhibited conformations and thermal stabilities similar to the wild-type peptide. In DNA-binding assays, the Lys mutant bound to target DNA, though its affinity was lower than that of the wild-type peptide. On the other hand, the Leu and Hgn mutants had no ability to bind DNA, despite the similarity in their secondary structures to the wild-type. Our results demonstrate that, as with the Leu residue, the aliphatic carbon side chain of this Arg residue plays a key role in the formation of a stable zinc finger domain, and its terminal guanidinium group appears to be essential for DNA binding mediated through both electrostatic interaction and hydrogen bonding with DNA phosphate backbone.
authors.affiliation: Faculty of Pharmaceutical Sciences, Doshisha Women's University, Koudo, Kyotanabe-Shi 610-0395, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Negi
authors.forename: Miki
authors.initials: M
authors.lastname: Imanishi
authors.forename: Maeko
authors.initials: M
authors.lastname: Sasaki
authors.forename: Kazuya
authors.initials: K
authors.lastname: Tatsutani
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
authors.forename: Yukio
authors.initials: Y
authors.lastname: Sugiura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D051766:Early Growth Response Protein 1 / Q000138:chemical synthesis* / Q000235:genetics; D006801:Humans; D007930:Leucine / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D055550:Protein Stability; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013816:Thermodynamics; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D001120:Arginine; D007930:Leucine