Ubiquitin-conjugating enzymes (E2) play a crucial role in the attachment of ubiquitin to proteins. Together with ubiquitin ligases (E3), they catalyze the transfer of ubiquitin (Ub) onto lysines with high chemoselectivity. A subfamily of E2s, including yeast Ubc6 and human Ube2J2, also mediates noncanonical modification of serines, but the structural determinants for this chemical versatility remain unknown. Using a combination of X-ray crystallography, molecular dynamics (MD) simulations, and reconstitution approaches, we have uncovered a two-layered mechanism that underlies this unique reactivity. A rearrangement of the Ubc6/Ube2J2 active site enhances the reactivity of the E2-Ub thioester, facilitating attack by weaker nucleophiles. Moreover, a conserved histidine in Ubc6/Ube2J2 activates a substrate serine by general base catalysis. Binding of RING-type E3 ligases further increases the serine selectivity inherent to Ubc6/Ube2J2, via an allosteric mechanism that requires specific positioning of the ubiquitin tail at the E2 active site. Our results elucidate how subtle structural modifications to the highly conserved E2 fold yield distinct enzymatic activity.

中文翻译：

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泛素结合酶 J2 家族泛素化中化学选择性的决定因素。


泛素偶联酶 （E2） 在泛素与蛋白质的结合中起着至关重要的作用。它们与泛素连接酶 （E3） 一起催化泛素 （Ub） 以高化学选择性转移到赖氨酸上。E2 的一个亚家族，包括酵母 Ubc6 和人 Ube2J2，也介导丝氨酸的非经典修饰，但这种化学多功能性的结构决定因素仍然未知。结合 X 射线晶体学、分子动力学 （MD） 模拟和重构方法，我们发现了这种独特反应性背后的两层机制。Ubc6/Ube2J2 活性位点的重排增强了 E2-Ub 硫酯的反应性，促进了较弱亲核试剂的攻击。此外，Ubc6/Ube2J2 中保守的组氨酸通过一般碱基催化激活底物丝氨酸。RING 型 E3 连接酶的结合进一步增加了 Ubc6/Ube2J2 固有的丝氨酸选择性，通过一种变构机制，该机制需要将泛素尾部特异性定位在 E2 活性位点。我们的结果阐明了对高度保守的 E2 折叠的微小结构修饰如何产生不同的酶活性。