Ai Huasong, Tong Zebin, Deng Zhiheng, Shi Qiang, Tao Shixian, Sun Gaoge, Liang Jiawei, Sun Maoshen, Wu Xiangwei, Zheng Qingyun, Liang Lujun, Yin Hang, Li Jia-Bin, Gao Shuai, Tian Changlin, Liu Lei, Pan Man
Institute of Translational Medicine, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
New Cornerstone Science Laboratory, Tsinghua-Peking Joint Center for Life Sciences, Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Center for Synthetic and Systems Biology, Department of Chemistry, Tsinghua University, Beijing, China.
Nat Chem Biol. 2025 May;21(5):668-680. doi: 10.1038/s41589-024-01750-x. Epub 2024 Oct 11.
The DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, has a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent K13 and K15 (K13/15) sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to site-specifically ubiquitinate H2A K13/15 has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5cUb (ubiquitin) in complex with nucleosomes. Here we developed chemical strategies and determined the cryo-electron microscopy structures of the RNF168-UbcH5cUb-nucleosome complex captured in transient H2A K13/15 monoubiquitination and adjacent dual monoubiquitination reactions, providing a 'helix-anchoring' mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the 'compass-binding' mode of dimeric E3 ligases. Our work not only provides structural snapshots of H2A K13/15 site-specific monoubiquitination and adjacent dual monoubiquitination but also offers a near-atomic-resolution structural framework for understanding pathogenic amino acid substitutions and physiological modifications of RNF168.
DNA损伤修复调节蛋白RNF168是一种单体环型E3连接酶，通过对H2A N端尾部相邻的K13和K15（K13/15）位点进行特异性高效泛素化，在调节细胞命运和DNA修复中起关键作用。然而，由于缺乏RNF168与UbcH5cUb（泛素）与核小体复合物的高分辨率结构，长期以来一直阻碍着人们对RNF168如何与同源E2酶UbcH5c协同作用以位点特异性泛素化H2A K13/15的理解。在这里，我们开发了化学策略，并确定了在瞬时H2A K13/15单泛素化和相邻双单泛素化反应中捕获的RNF168-UbcH5cUb-核小体复合物的冷冻电镜结构，为单体E3连接酶RNF168在核小体上提供了一种“螺旋锚定”模式，这与二聚体E3连接酶的“罗盘结合”模式形成对比。我们的工作不仅提供了H2A K13/15位点特异性单泛素化和相邻双单泛素化的结构快照，还为理解RNF168的致病性氨基酸取代和生理修饰提供了一个近原子分辨率的结构框架。
