Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, China.
Center of Medical Physics and Technology, Hefei Institute of Physical Science, Chinese Academy of Science, Hefei, China.
FEBS J. 2018 Jun;285(11):2091-2103. doi: 10.1111/febs.14469. Epub 2018 Apr 24.
As a reader of di-methylated arginine on various proteins, such as histone, RNA polymerase II, PIWI and Fragile X mental retardation protein, the Tudor domain of Tudor domain-containing protein 3 (TDRD3) mediates transcriptional activation in nucleus and formation of stress granules in the cytoplasm. Despite the TDRD3 implication in cancer cell proliferation and invasion, warheads to block the di-methylated arginine recognition pocket of the TDRD3 Tudor domain have not yet been uncovered. Here we identified 14 small molecule hits against the TDRD3 Tudor domain through NMR fragment-based screening. These hits were further cross-validated by using competitive fluorescence polarization and isothermal titration calorimetry experiments. The crystal structure of the TDRD3 Tudor domain in complex with hit 1 reveals a distinct binding mode from the nature substrate. Hit 1 protrudes into the aromatic cage of the TDRD3 Tudor domain, where the aromatic residues are tilted to accommodate a sandwich-like π-π interaction. The side chain of the conserved residue N596 swings away 3.1 Å to form a direct hydrogen bond with hit 1. Moreover, this compound shows a decreased affinity against the single Tudor domain of survival motor neuron protein, but no detectable binding to neither the tandem Tudor domain of TP53-binding protein 1 nor the extended Tudor domain of staphylococcal nuclease domain-containing protein 1. Our work depicts the structural plasticity of the TDRD3 Tudor domain and paves the way for the subsequent structure-guided discovery of selective inhibitors targeting Tudor domains.
Structural data are available in the PDB under the accession number 5YJ8.
作为各种蛋白质（如组蛋白、RNA 聚合酶 II、PIWI 和脆性 X 智力迟钝蛋白）上二甲基精氨酸的读取器，Tudor 结构域包含蛋白 3（TDRD3）的 Tudor 结构域介导核内转录激活和细胞质中应激颗粒的形成。尽管 TDRD3 参与癌细胞增殖和侵袭，但尚未发现阻止 TDRD3 Tudor 结构域中二甲基精氨酸识别口袋的弹头。在这里，我们通过 NMR 基于片段的筛选鉴定了针对 TDRD3 Tudor 结构域的 14 个小分子命中。这些命中进一步通过竞争性荧光偏振和等温滴定量热法实验进行交叉验证。与命中 1 结合的 TDRD3 Tudor 结构域的晶体结构揭示了一种与天然底物不同的结合模式。命中 1 突出到 TDRD3 Tudor 结构域的芳香笼中，其中芳香残基倾斜以容纳三明治样的 π-π 相互作用。保守残基 N596 的侧链摆动 3.1 Å 与命中 1 形成直接氢键。此外，该化合物对生存运动神经元蛋白的单个 Tudor 结构域的亲和力降低，但对 TP53 结合蛋白 1 的串联 Tudor 结构域或葡萄球菌核酸酶结构域包含蛋白 1 的扩展 Tudor 结构域均无检测到结合。我们的工作描绘了 TDRD3 Tudor 结构域的结构可塑性，并为随后针对 Tudor 结构域的选择性抑制剂的结构指导发现铺平了道路。
结构数据可在 PDB 中以 5YJ8 的访问号获得。
