Institut National de Santé et de Recherche Médicale U964/Centre National de Recherche Scientifique UMR 7104/Université de Strasbourg, Illkirch, France.
PLoS One. 2011;6(11):e27599. doi: 10.1371/journal.pone.0027599. Epub 2011 Nov 11.
The human UHRF1 protein (ubiquitin-like containing PHD and RING finger domains 1) has emerged as a potential cancer target due to its implication in cell cycle regulation, maintenance of DNA methylation after replication and heterochromatin formation. UHRF1 functions as an adaptor protein that binds to histones and recruits histone modifying enzymes, like HDAC1 or G9a, which exert their action on chromatin. In this work, we show the binding specificity of the PHD finger of human UHRF1 (huUHRF1-PHD) towards unmodified histone H3 N-terminal tail using native gel electrophoresis and isothermal titration calorimetry. We report the molecular basis of this interaction by determining the crystal structure of huUHRF1-PHD in complex with the histone H3 N-terminal tail. The structure reveals a new mode of histone recognition involving an extra conserved zinc finger preceding the conventional PHD finger region. This additional zinc finger forms part of a large surface cavity that accommodates the side chain of the histone H3 lysine K4 (H3K4) regardless of its methylation state. Mutation of Q330, which specifically interacts with H3K4, to alanine has no effect on the binding, suggesting a loose interaction between huUHRF1-PHD and H3K4. On the other hand, the recognition appears to rely on histone H3R2, which fits snugly into a groove on the protein and makes tight interactions with the conserved aspartates D334 and D337. Indeed, a mutation of the former aspartate disrupts the formation of the complex, while mutating the latter decreases the binding affinity nine-fold.
人类 UHRF1 蛋白（泛素样结构域包含 PH 和 RING 指结构域 1）已成为潜在的癌症靶点，因为其参与细胞周期调控、复制后 DNA 甲基化的维持和异染色质形成。UHRF1 作为一种衔接蛋白，可与组蛋白结合并招募组蛋白修饰酶，如 HDAC1 或 G9a，这些酶在染色质上发挥作用。在这项工作中，我们使用天然凝胶电泳和等温滴定量热法显示了人 UHRF1（huUHRF1-PHD）的 PH 结构域对未修饰的组蛋白 H3 N 端尾巴的结合特异性。我们通过确定 huUHRF1-PHD 与组蛋白 H3 N 端尾巴复合物的晶体结构报告了这种相互作用的分子基础。结构揭示了一种新的组蛋白识别模式，涉及在传统 PH 结构域区域之前的额外保守锌指。这个额外的锌指形成了一个大的表面腔的一部分，容纳了组蛋白 H3 赖氨酸 K4（H3K4）的侧链，无论其甲基化状态如何。突变与 H3K4 特异性相互作用的 Q330 为丙氨酸对结合没有影响，这表明 huUHRF1-PHD 与 H3K4 之间的相互作用比较松散。另一方面，这种识别似乎依赖于组蛋白 H3R2，它紧密地适合于蛋白质上的一个凹槽，并与保守的天冬氨酸 D334 和 D337 紧密相互作用。事实上，前一个天冬氨酸的突变会破坏复合物的形成，而后者的突变则会使结合亲和力降低九倍。
