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.
Keywords: MUL1; MUL1-RING; RING domain; Ube2D2; crystallization; mitochondrial E3 ubiquitin ligase 1; ubiquitylation.