Tong Zebin, Wu Xiangwei, Cai Hongyi, Wu Shidian, Zhang Tianyi, Deng Zhiheng, Xu Ziyu, Yuan Rujing, Ai Huasong, Liu Lei, Pan Man
Institute of Translational Medicine, School of Pharmaceutical Sciences, School of Chemistry and Chemical Engineering, National Center for Translational Medicine (Shanghai), Shanghai Key Laboratory for Antibody-Drug Conjugates with Innovative Target, 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 Aug 15. doi: 10.1038/s41589-025-01985-2.
E4 enzymes amplify and remodel ubiquitin chain signals beyond the conventional E1-E2-E3 cascade. The first identified E4 enzyme Ufd2 preferentially catalyzes K48/K29 branched ubiquitin chains, yet the structural mechanism remains unknown. Here, we combined chemical biology and cryo-electron microscopy to visualize stable intermediates in Ufd2 loading ubiquitin at K48 of proximal ubiquitin on K29-linked di- and triubiquitin. Our data reveal that the core region of Ufd2 functions as an unprecedented K29 diubiquitin binding domain, interacting extensively with proximal and distal ubiquitin, which orients the K48 site of proximal ubiquitin toward the active site of Ubc4, facilitating K48/K29 branched ubiquitin chain formation. We also identified a unique dimeric conformation where dimerized Ufd2 and Ubc4 stabilize each other's distal ubiquitin during branching on K29 triubiquitin. Our findings provide mechanistic insights into the assembly of K48/K29 branched ubiquitin chains by the E4 enzyme Ufd2 and highlight the spatial cooperation among multiple pairs of ubiquitin-related enzymes on longer ubiquitin chains.
E4酶能在传统的E1-E2-E3级联反应之外放大并重塑泛素链信号。首个被鉴定出的E4酶Ufd2优先催化K48/K29分支泛素链，但结构机制仍不清楚。在此，我们结合化学生物学和冷冻电子显微镜技术，以可视化Ufd2在K29连接的二聚泛素和三聚泛素上近端泛素的K48位点加载泛素时的稳定中间体。我们的数据表明，Ufd2的核心区域作为一个前所未有的K29二聚泛素结合结构域，与近端和远端泛素广泛相互作用，将近端泛素的K48位点导向Ubc4的活性位点，促进K48/K29分支泛素链的形成。我们还鉴定出一种独特的二聚体构象，在K29三聚泛素分支过程中，二聚化的Ufd2和Ubc4相互稳定彼此的远端泛素。我们的研究结果为E4酶Ufd2组装K48/K29分支泛素链提供了机制见解，并突出了在更长的泛素链上多对泛素相关酶之间的空间协作。
