Department of Chemistry and Research Institute of Natural Science, Gyeongsang National University, Jinju, Gyeongnam 660-701, Republic of Korea.
Department of Chemistry, KAIST, Daejeon 305-701, Republic of Korea.
Arch Biochem Biophys. 2014 Sep 15;558:95-103. doi: 10.1016/j.abb.2014.06.026. Epub 2014 Jul 8.
The Zα domains of human ADAR1 (ZαADAR1) bind to Z-DNA via interaction mediated by the α3-core and β-hairpin. Five residues in the α3 helix and four residues in the β-hairpin play important roles in Zα function, forming direct or water-mediated hydrogen bonds with DNA backbone phosphates or interacting hydrophobically with DNA bases. To understand the roles of these residues during B-Z transition of duplex DNA, we performed NMR experiments on complexes of various ZαADAR1 mutants with a 6-bp DNA duplex at various protein-to-DNA molar ratios. Our study suggests that single mutations at residues K169, N173, or Y177 cause unusual conformational changes in the hydrophobic faces of helices α1, α2, and α3, which dramatically decrease the Z-DNA binding affinity. 1D imino proton spectra and chemical shift perturbation showed that single mutations at residues K170, R174, T191, P192, P193, or W195 slightly affected the Z-DNA binding affinity. A hydrogen exchange study proved that the K170A- and R174A-ZαADAR1 proteins could efficiently change B-DNA to left-handed Z-DNA via an active B-Z transition pathway, whereas the G2·C5 base pair was significantly destabilized compared to wild-type ZαADAR1.
人源 ADAR1 的 Zα 结构域（ZαADAR1）通过α3 核心和β发夹介导的相互作用与 Z-DNA 结合。α3 螺旋中的 5 个残基和β发夹中的 4 个残基在 Zα 功能中发挥重要作用，与 DNA 骨架磷酸形成直接或水介导的氢键，或与 DNA 碱基疏水相互作用。为了了解这些残基在双链 DNA 的 B-Z 转变过程中的作用，我们在不同蛋白与 DNA 摩尔比下，对各种 ZαADAR1 突变体与 6 个碱基对 DNA 双链体的复合物进行了 NMR 实验。我们的研究表明，残基 K169、N173 或 Y177 的单点突变导致α1、α2 和α3 螺旋的疏水面发生异常构象变化，从而极大地降低了 Z-DNA 的结合亲和力。1D 亚氨基质子谱和化学位移扰动表明，残基 K170、R174、T191、P192、P193 或 W195 的单点突变对 Z-DNA 的结合亲和力略有影响。氢交换研究证明，K170A 和 R174A-ZαADAR1 蛋白可以通过有效的 B-Z 转变途径将 B-DNA 高效地转变为左手 Z-DNA，而与野生型 ZαADAR1 相比，G2·C5 碱基对明显不稳定。
