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
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
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DOI：
10.1021/bi200697p
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发表时间：
2011-07-19
期刊：
影响因子：
2.9
通讯作者：
Sugiura, Yukio
中科院分区：
文献类型：
--
作者：
Negi, Shigeru;Imanishi, Miki;Sugiura, Yukio
Cys（2）His（2）型锌指是一种常见的DNA结合基序，广泛应用于人工锌指蛋白的设计。在几乎所有的Cys（2）His（2）型锌指中，α-螺旋DNA识别位点的第4位被参与形成最小疏水核心的Leu残基占据。然而，天然Zif 268的第三锌指结构域含有Arg残基而不是保守的Leu。我们在本研究中的目的是澄清的作用，这精氨酸在形成一个稳定的结构域结构和DNA结合取代它与赖氨酸，亮氨酸，或Hgn，具有不同的末端侧链结构。评估的金属结合性能，肽构象，和DNA结合能力的突变体。所有三种突变指3肽表现出类似于野生型肽的构象和热稳定性。在DNA结合试验中，赖氨酸突变体与靶DNA结合，尽管其亲和力低于野生型肽。另一方面，Leu和Hgn突变体没有结合DNA的能力，尽管它们的二级结构与野生型相似。我们的研究结果表明，与Leu残基一样，该Arg残基的脂肪族碳侧链在形成稳定的锌指结构域中起着关键作用，其末端胍基似乎是通过静电相互作用和与DNA磷酸骨架的氢键作用介导的DNA结合所必需的。
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 a-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 guaniclinium group appears to be essential for DNA binding mediated through both electrostatic interaction and hydrogen bonding with DNA phosphate backbone.