Page 22 - U. Protein structure and function

[U. Protein structure and function-17]

Solution structure of MUL1-RING domain and its interaction

with p53 transactivation domain

Min-Sung Lee¹˙²˙#, Sang-Ok Lee¹˙⁶, Mi-Kyung Lee¹, Gwan-Su Yi⁵, Chong-Kil Lee⁶, Kyoung-Seok Ryu³˙⁴˙*,
Seung-Wook Chi¹˙²˙*

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

Korea, ²Department of Proteome Structural Biology, KRIBB School of Bioscience, University of Science and
Technology, Daejeon 34113, Republic of Korea, ³Department of Bio-Analytical Science, University of Science and
Technology, Daejeon 34113, Republic of Korea, ⁴Protein Structure Research Group, Korea Basic Science Institute,

Cheongju-si 28119, Republic of Korea, ⁵Department of Bio and Brain Engineering, KAIST, Daejeon 34141, Republic

of Korea, ⁶College of Pharmacy, Chungbuk National University, Cheongju 28644, Republic of Korea

Mitochondrial E3 ubiquitin ligase 1 (MUL1) is a multifunctional mitochondrial protein involved in various biological
processes such as mitochondrial dynamics, cell growth, apoptosis, and mitophagy. MUL1 mediates the ubiquitylation

of mitochondrial p53 for proteasomal degradation. Although the interaction of MUL1-RING domain with its
substrate, p53, is a unique mechanism in RING-mediated ubiquitylation, the molecular basis of this process remains

unknown. In this study, we determined the solution structure of the MUL1-RING domain and characterized its
interaction with the p53 transactivation domain (p53-TAD) by NMR spectroscopy. The overall structure of the MUL1-

RING domain is similar to those of RING domains of other E3 ubiquitinases. The MUL1-RING domain adopts a bbab
fold with three anti-parallel b-strands and one a-helix, containing a canonical cross-brace motif for the ligation of

two zinc ions. Through NMR chemical shift perturbation experiments, we determined the p53-TAD-binding site in
the MUL1-RING domain and showed that the MUL1-RING domain interacts mainly with the p53-TAD2 subdomain

composed of residues 39e57. Taken together, our results provide a molecular basis for the novel recognition
mechanism of the p53-TAD substrate by the MUL1-RING domain.