Mitochondrial E3 ubiquitin ligase 1 (MUL1) is located in the mitochondrial outer membrane and regulates various biological processes, including apoptosis, cell growth, mitophagy and mitochondrial dynamics. The C-terminal region of MUL1 faces the cytoplasm and contains the RING domain (MUL1-RING) where the Ub~E2 thioester binds. Unlike most RING-type E3 enzymes, MUL1-RING alone does not have an additional region that recruits a substrate protein, yet is still able to ubiquitylate the substrate, the p53 protein. Nevertheless, the exact mechanism of the ubiquitylation of p53 by MUL1-RING has not yet been elucidated. In order to understand this novel ubiquitylation mechanism, it is necessary to determine the three-dimensional structures of MUL1-RING and of its complex with the cognate E2 enzyme. Here, Ube2D2 was validated as a functional E2 enzyme for the ubiquitylation of the p53 transactivation domain (p53-TAD) by MUL1-RING, and purification and crystallization processes for MUL1-RING and the MUL1-RING-Ube2D2 complex are reported.

中文翻译：

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线粒体 E3 泛素连接酶 1 的 RING 结构域及其与 Ube2D2 的复合物：结晶和 X 射线衍射。


线粒体 E3 泛素连接酶 1 (MUL1) 位于线粒体外膜，调节多种生物过程，包括细胞凋亡、细胞生长、线粒体自噬和线粒体动力学。 MUL1 的 C 末端区域面向细胞质，并包含 Ub~E2 硫酯结合的 RING 结构域 (MUL1-RING)。与大多数 RING 型 E3 酶不同，MUL1-RING 本身不具有招募底物蛋白的额外区域，但仍然能够泛素化底物 p53 蛋白。然而，MUL1-RING 泛素化 p53 的确切机制尚未阐明。为了理解这种新颖的泛素化机制，有必要确定 MUL1-RING 及其与同源 E2 酶复合物的三维结构。在此，Ube2D2 被验证为一种功能性 E2 酶，可通过 MUL1-RING 泛素化 p53 反式激活结构域 (p53-TAD)，并报告了 MUL1-RING 和 MUL1-RING-Ube2D2 复合物的纯化和结晶过程。