Page 24 - U. Protein structure and function

[U. Protein structure and function-18]

The RING domain of mitochondrial E3 ubiquitin ligase 1 and

its complex with Ube2D2: crystallization and X-ray diffraction

Sang-Ok Lee¹˙²˙#, Chong-Kil Lee², Kyoung-Seok Ryu³˙⁴˙*, Seung-Wook Chi¹˙⁵˙*

¹Disease Target Structure Research Center, Division of Biomedical Research , KRIBB, Daejeon 34141, Republic of

Korea, ²College of Pharmacy, Chungbuk National University, Cheongju 28644, Republic of Korea, ³Protein Structure
Research Group, Korea Basic Science Institute, Cheongju-si 28119, Republic of Korea, ⁴Department of Bio-

Analytical Science, KBSI School of Bio-Analytical Science, University of Sciecne and Technology, Daejeon 34113,
Republic of Korea, ⁵Department of Proteome Structural Biology, KRIBB School of Bioscience, University of Sciecne

and Technology, Daejeon 34113, Republic of Korea

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.