Synthetic E2-Ub-nucleosome conjugates for studying nucleosome ubiquitination
Synthetic E2-Ub-nucleosome conjugates for studying nucleosome ubiquitination
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DOI：
10.1016/j.chempr.2023.01.012
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发表时间：
2023-05-11
期刊：
影响因子：
23.5
通讯作者：
Liu, Lei
中科院分区：
文献类型：
--
作者：
Ai, Huasong;Tong, Zebin;Liu, Lei
e2 -泛素（Ub）缀合物是研究E3机制的有效化学生物学工具，但迄今为止，报道的系统要么没有底物，要么只有短肽底物。在这里，我们描述了E2-Ub-核小体缀合物的实际化学合成，该缀合物可以捕获E3介导的Ub从E2转移到核小体的瞬态中间体，形成稳定的E3/ E2/核小体复合物，其表征和结构测定揭示了不同的E3-E2模块如何建立特定的结构，将Ub定向到目标赖氨酸。我们的工作提供了第一个例子来支持使用化学捕获策略来研究全长和折叠蛋白上E3介导的ub转移机制，并克服了线性E3/E2融合策略可能存在的局限性，即头尾融合可能妨碍E3和/或E2与底物的最佳相互作用，并且当E3由多个亚基组成时，很难描述E2头尾融合的位置。
E2-ubiquitin (Ub) conjugates are useful chemical biology tools for studying the E3 mechanisms, but so far, the reported systems have involved either no substrates or only short peptide substrates. Here, we describe a practical chemical synthesis of E2-Ub-nucleo-some conjugates that can trap the transient intermediates of E3 -mediated Ub transfer from E2 to nucleosomes to form stable E3/ E2/nucleosome complexes, whose characterization and structure determination revealed how distinct E3-E2 modules establish a spe-cific architecture to orient the Ub toward the target lysine. Our work provides the first example to support the use of a chemical trapping strategy to study the E3-mediated Ub-transferring mechanisms on full-length and folded proteins and overcomes the possible limita-tions of the linear E3/E2 fusion strategy-that head-to-tail fusion may preclude the optimal interaction of E3 and/or E2 with the substrate and that delineation of where E2 is head-to-tail fused is difficult when E3 is composed of multiple subunits.