Structural basis for transthiolation intermediates in the ubiquitin pathway

• 成果类型: Article
• 署名作者: Kochanczyk, Tomasz; Hann, Zachary S.; Lux, Michaelyn C.; Delos Reyes, Avelyn Mae V.; Ji, Cheng; Tan, Derek S.; Lima, Christopher D.
• 署名单位: Memorial Sloan Kettering Cancer Center; Howard Hughes Medical Institute; Memorial Sloan Kettering Cancer Center; Memorial Sloan Kettering Cancer Center; Cornell University; Memorial Sloan Kettering Cancer Center
• 刊物名称: Nature
• ISSN/ISSBN: 0028-3690
• DOI: 10.1038/s41586-024-07828-9
• 发表日期: 2024-09-05
• 关键词: beam-induced motion; e3 ligase; bayesian-approach; insights; binding; mechanism; reveals; Visualization; substrate; complex

摘要

Transthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins(1-3). For the Ub pathway, E1 enzymes catalyse transthiolation from an E1 similar to Ub thioester to an E2 similar to Ub thioester. Transthiolation is also required for transfer of Ub from an E2 similar to Ub thioester to HECT (homologous to E6AP C terminus) and RBR (ring-between-ring) E3 ligases to form E3 similar to Ub thioesters(4-6). How isoenergetic transfer of thioester bonds is driven forward by enzymes in the Ub pathway remains unclear. Here we isolate mimics of transient transthiolation intermediates for E1-Ub(T)-E2 and E2-Ub(T)-E3(HECT) complexes (where T denotes Ub in a thioester or Ub undergoing transthiolation) using a chemical strategy with native enzymes and near-native Ub to capture and visualize a continuum of structures determined by single-particle cryo-electron microscopy. These structures and accompanying biochemical experiments illuminate conformational changes in Ub, E1, E2 and E3 that are coordinated with the chemical reactions to facilitate directional transfer of Ub from each enzyme to the next.