Mouse Piwi interactome identifies binding mechanism of Tdrkh Tudor domain to arginine methylated Miwi
Mouse Piwi interactome identifies binding mechanism of Tdrkh Tudor domain to arginine methylated Miwi
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DOI：
10.1073/pnas.0911640106
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发表时间：
2009-12-01
影响因子：
11.1
通讯作者：
Pawson, Tony
中科院分区：
文献类型：
--
作者：
Chen, Chen;Jin, Jing;Pawson, Tony
都铎结构域是介导蛋白质相互作用的蛋白质模块，可能通过与甲基化配体结合。以果蝇Tudor及其哺乳动物同系物Tdrd1、Tdrd4/RNF17和Tdrd6为代表的一组种系特异性单和多Tudor结构域蛋白（TDRDs）在生发颗粒/幼体形成和生殖细胞的规范和分化中发挥着进化保守的作用。然而，它们的生理配体以及配体识别的生化和结构基础在很大程度上是不清楚的。本研究通过内源性小鼠Piwi蛋白（Miwi和Mili）的免疫沉淀和piRNA通路相关复合物的蛋白质组学分析，我们发现Tudor蛋白的TDRD组是Piwi家族蛋白的生理结合伙伴。此外，质谱分析表明，Miwi和Mili的n端RG重复序列中的精氨酸残基在体内被甲基化。值得注意的是，我们发现Tdrkh/Tdrd2，一个在Miwi复合体中发现的新的单Tudor结构域蛋白，在男性生殖细胞的细胞质中表达，并与Miwi直接相关。诱变研究将Miwi-Tdrkh相互作用定位到Miwi的n端RG/RA重复序列，并表明Tdrkh Tudor结构域对结合至关重要。此外，我们还解决了Tdrkh Tudor结构域的晶体结构，揭示了适合甲基化精氨酸的芳香结合袋和带负电荷的结合表面。我们的研究结果确定了生殖细胞中由种系Tudor结构域和甲基化Piwi家族蛋白介导的甲基化导向的蛋白相互作用机制，并提出了在piRNA沉默通路中调节Piwi蛋白组织和功能的复杂模式。
Tudor domains are protein modules that mediate protein-protein interactions, potentially by binding to methylated ligands. A group of germline specific single and multiTudor domain containing proteins (TDRDs) represented by drosophila Tudor and its mammalian orthologs Tdrd1, Tdrd4/RNF17, and Tdrd6 play evolutionarily conserved roles in germinal granule/nuage formation and germ cell specification and differentiation. However, their physiological ligands, and the biochemical and structural basis for ligand recognition, are largely unclear. Here, by immunoprecipitation of endogenous murine Piwi proteins (Miwi and Mili) and proteomic analysis of complexes related to the piRNA pathway, we show that the TDRD group of Tudor proteins are physiological binding partners of Piwi family proteins. In addition, mass spectrometry indicates that arginine residues in RG repeats at the N-termini of Miwi and Mili are methylated in vivo. Notably, we found that Tdrkh/Tdrd2, a novel single Tudor domain containing protein identified in the Miwi complex, is expressed in the cytoplasm of male germ cells and directly associates with Miwi. Mutagenesis studies mapped the Miwi-Tdrkh interaction to the very N-terminal RG/RA repeats of Miwi and showed that the Tdrkh Tudor domain is critical for binding. Furthermore, we have solved the crystal structure of the Tdrkh Tudor domain, which revealed an aromatic binding pocket and negatively charged binding surface appropriate for accommodating methylated arginine. Our findings identify a methylation-directed protein interaction mechanism in germ cells mediated by germline Tudor domains and methylated Piwi family proteins, and suggest a complex mode of regulating the organization and function of Piwi proteins in piRNA silencing pathways.