Identification of a catalytic active but non-aggregating MDM2 RING domain variant

Magnussen, H. M. and Huang, D. T. (2021) Identification of a catalytic active but non-aggregating MDM2 RING domain variant. Journal of Molecular Biology, 433(5), 166807. (doi: 10.1016/j.jmb.2021.166807) (PMID:33450248) (PMCID:PMC7895813)

Abstract

As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2's ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)-Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors.

• Item Type: Articles
• Additional Information: The authors would like to thank Core Services and Advanced Technologies at the Cancer Research UK Beatson Institute (C596/A17196), Catherine Winchester for critical reading of the manuscript, and Diamond Light Source for access to stations I03, I04 and I04-1 (BAG allocation mx16258). This work was supported by the Beatson Institute’s Cancer Research UK core funding (A17196), by Cancer Research UK (A29256) and the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement n° 647849)..
• Keywords: E3, MDM2, Ubiquitin ligase, aggregation, protein design.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Huang, Professor Danny and Magnussen, Helge

Creator Roles

Magnussen, H. M.: Conceptualization, Methodology, Investigation
Huang, D. T.: Conceptualization, Supervision, Funding acquisition

• Authors: Magnussen, H. M., and Huang, D. T.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: Journal of Molecular Biology
• Publisher: Elsevier
• ISSN: 0022-2836
• ISSN (Online): 1089-8638
• Published Online: 13 January 2021
• Copyright Holders: Copyright © 2021 The Authors
• First Published: First published in Journal of Molecular Biology 433(5): 166807
• Publisher Policy: Reproduced under a Creative Commons License