Ufmylation, a protein modification by Ubiquitin-like (UBL) protein UFM1, plays a crucial role in several cellular processes including DNA damage response, protein translation and ER homeostasis. To date, little is known how the enzymes responsible for this modification coordinate their action. Here we have studied the details of UFL1 (E3) activity, its binding to UFC1 (E2), and its relation to UBA5 (E1), using a combination of structural modeling with Alphafold2, X-ray crystallography, NMR, and in vitro biochemical activity assays. Guided by an Alphafold2 model, we generated an active UFL1 fusion construct that includes its cofactor DDRGK1, and solved the first crystal structure of this critical interaction. This fusion construct also unveiled the importance of the N-terminal helix of UFL1 for its binding to UFC1, which was validated by ITC and NMR experiments. Importantly, the binding site suggested by our structural model of the UFL1-UFC1 interaction reveals a conserved interface, and suggests a competition for binding to UFC1 between UFL1 and UBA5, which we reconfirmed by NMR. Altogether, our study reveals a novel, terminal helix-mediated regulatory mechanism which coordinates the cascade of E1-E2-E3 mediated transfer of UFM1 to its substrate, and provides new leads to target this important modification.

中文翻译：

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UFL1-UFC1 相互作用的结构研究揭示了 UFL1 N 端螺旋对 ufmylation 的重要性

Ufmylation 是一种由泛素样 (UBL) 蛋白 UFM1 修饰的蛋白质，在多种细胞过程中起着至关重要的作用，包括 DNA 损伤反应、蛋白质翻译和 ER 稳态。迄今为止，很少有人知道负责这种修饰的酶如何协调它们的作用。在这里，我们研究了 UFL1 (E3) 活动的细节、它与 UFC1 (E2) 的结合以及它与 UBA5 (E1) 的关系，结合使用结构建模与 Alphafold2、X 射线晶体学、NMR 和体外生化活性测定。在 Alphafold2 模型的指导下，我们生成了一个活跃的 UFL1 融合结构，其中包括其辅因子 DDRGK1，并解决了这一关键相互作用的第一个晶体结构。这种融合构建体还揭示了 UFL1 的 N 端螺旋对于其与 UFC1 结合的重要性，这已通过 ITC 和 NMR 实验验证。重要的是，我们的 UFL1-UFC1 相互作用结构模型建议的结合位点揭示了一个保守的界面，并表明 UFL1 和 UBA5 之间竞争结合 UFC1，我们通过 NMR 再次确认了这一点。总而言之，我们的研究揭示了一种新的末端螺旋介导的调节机制，它协调 E1-E2-E3 介导的 UFM1 转移到其底物的级联，并提供了针对这一重要修饰的新线索。