Shi Qiang, Deng Zhiheng, Zhang Liying, Tong Zebin, Li Jia-Bin, Chu Guo-Chao, Ai Huasong, Liu Lei
New Cornerstone Science Laboratory, Tsinghua-Peking Joint Center for Life Sciences, MOE Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Center for Synthetic and Systems Biology, Department of Chemistry, Tsinghua University, Beijing, 100084, China.
College of Pharmaceutical Sciences, Soochow University, Suzhou, 215031, China.
Angew Chem Int Ed Engl. 2025 Jan 2;64(1):e202413651. doi: 10.1002/anie.202413651. Epub 2024 Nov 6.
The chemical synthesis of histones with homogeneous modifications is a powerful approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). In this study, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on H1 ubiquitylated chromatosomes with different ubiquitin chain lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and the chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally defined polyubiquitylated H1.0 by the CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.
化学合成具有均一修饰的组蛋白是定量解析不同翻译后修饰（PTM）之间功能串扰的有力方法。在本研究中，我们开发了一种便捷的位点特异性（多）泛素化策略（CAEPL，半胱氨酸氨基乙基化与酶促蛋白质连接相结合），该策略将半胱氨酸氨基乙基化反应与泛素激活酶UBA1辅助的天然化学连接过程整合在一起。利用该策略，我们成功制备了单泛素化以及K63连接的双泛素化和三泛素化的连接组蛋白H1.0蛋白，并将其整合到单个染色质小体中。对具有不同泛素链长度的H1泛素化染色质小体上的不同RNF168构建体进行定量生化分析表明，K63连接的多泛素化H1.0可通过增强RNF168与染色质小体之间的亲和力直接刺激RNF168的泛素化活性。随后对RNF168/UbcH5c-Ub/H1.0-K63-Ub染色质小体复合物进行冷冻电镜结构分析，揭示了RNF168 UDM1结构域与H1.0上K63连接的泛素链之间潜在的招募方向。最后，我们在不对称的H1.0-K63-Ub双核小体底物背景下探究了H1.0泛素化对RNF168活性的影响，揭示了核小体间和核小体内串扰具有相当的刺激作用。总体而言，我们的研究突出了通过CAEPL策略获得结构明确的多泛素化H1.0的重要性，从而能够深入开展连接组蛋白H1.0与核心组蛋白H2A泛素化之间的翻译后修饰串扰的机制研究。
