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^1,2,3. For the Ub pathway, E1 enzymes catalyse transthiolation from an E1~Ub thioester to an E2~Ub thioester. Transthiolation is also required for transfer of Ub from an E2~Ub thioester to HECT (homologous to E6AP C terminus) and RBR (ring-between-ring) E3 ligases to form E3~Ub thioesters^4,5,6. 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.

转硫基化（也称为转硫酯化）反应用于乙酰辅酶 A、脂肪酸和聚酮化合物的生物合成，以及泛素 (Ub) 和泛素样 (Ubl) 蛋白的翻译后修饰^1,2,3 。对于Ub途径，E1酶催化从E1~Ub硫酯到E2~Ub硫酯的转硫作用。将 Ub 从 E2~Ub 硫酯转移至 HECT（与 E6AP C 末端同源）和 RBR（环间环）E3 连接酶以形成 E3~Ub 硫酯^4,5,6也需要转硫基作用。 Ub 途径中的酶如何驱动硫酯键的等能转移仍不清楚。在这里，我们使用天然酶和附近的化学策略分离了 E1–Ub(T)–E2 和 E2–Ub(T)–E3 ^HECT复合物的瞬时转硫基中间体的模拟物（其中 T 表示硫酯中的 Ub 或正在进行转硫基的 Ub）。 -原生 Ub 用于捕获和可视化由单粒子冷冻电子显微镜确定的连续结构。这些结构和伴随的生化实验阐明了 Ub、E1、E2 和 E3 的构象变化，这些变化与化学反应相协调，以促进 Ub 从每种酶到下一种酶的定向转移。