Kumar Manoj, Banerjee Sayanika, Cohen-Kfir Einav, Mitelberg Marissa Basia, Tiwari Suryakant, Isupov Michail N, Dessau Moshe, Wiener Reuven
Department of Biochemistry and Molecular Biology, The Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel.
Azrieli Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, 1311502, Israel.
Nat Commun. 2025 Apr 25;16(1):3912. doi: 10.1038/s41467-025-58826-y.
The conjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBL) to target proteins is a crucial post-translational modification that typically involves nucleophilic attack by a lysine on a charged E2 enzyme (E2~Ub/UBL), forming an oxyanion intermediate. Stabilizing this intermediate through an oxyanion hole is vital for progression of the reaction. Still, the mechanism of oxyanion stabilization in E2 enzymes remains unclear, although an asparagine residue in the conserved HPN motif of E2 enzymes was suggested to stabilize the oxyanion intermediate. Here, we study the E2 enzyme UFC1, which presents a TAK rather than an HPN motif. Crystal structures of UFC1 mutants, including one that mimics the oxyanion intermediate, combined with in vitro activity assays, suggest that UFC1 utilizes two distinct types of oxyanion holes, one that stabilizes the oxyanion intermediate during trans-ufmylation mediated by the E3 ligase, and another that stabilizes cis-driven auto-ufmylation. Our findings indicate that oxyanion stabilization is influenced by multiple factors, including C-alpha hydrogen bonding, and is adaptable, enabling different modes of action.
泛素（Ub）或泛素样蛋白（UBL）与靶蛋白的缀合是一种关键的翻译后修饰，通常涉及赖氨酸对带电荷的E2酶（E2~Ub/UBL）进行亲核攻击，形成氧阴离子中间体。通过氧阴离子洞稳定该中间体对于反应的进行至关重要。尽管有人提出E2酶保守HPN基序中的一个天冬酰胺残基可稳定氧阴离子中间体，但E2酶中氧阴离子稳定的机制仍不清楚。在此，我们研究了呈现TAK基序而非HPN基序的E2酶UFC1。UFC1突变体的晶体结构，包括一个模拟氧阴离子中间体的突变体，结合体外活性测定，表明UFC1利用两种不同类型的氧阴离子洞，一种在E3连接酶介导的转Ufmylation过程中稳定氧阴离子中间体，另一种稳定顺式驱动的自身Ufmylation。我们的研究结果表明，氧阴离子的稳定受多种因素影响，包括C-α氢键，并且具有适应性，能够实现不同的作用模式。
