PUBLICATION
Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant
- Authors
- Magnussen, H.M., Huang, D.T.
- ID
- ZDB-PUB-210507-21
- Date
- 2021
- Source
- Journal of molecular biology 433: 166807 (Journal)
- Registered Authors
- Keywords
- Aggregation, E3, MDM2, Protein design, Ubiquitin ligase
- MeSH Terms
-
- Amino Acid Sequence
- Animals
- Biocatalysis
- Catalytic Domain
- Conserved Sequence
- Crystallography, X-Ray
- Gene Expression
- Humans
- Mammals
- Models, Molecular
- Protein Aggregates
- Protein Binding
- Protein Conformation, alpha-Helical
- Protein Conformation, beta-Strand
- Protein Domains
- Protein Interaction Domains and Motifs
- Protein Processing, Post-Translational*
- Proto-Oncogene Proteins c-mdm2/chemistry*
- Proto-Oncogene Proteins c-mdm2/genetics
- Proto-Oncogene Proteins c-mdm2/metabolism
- Recombinant Proteins/chemistry
- Recombinant Proteins/genetics
- Recombinant Proteins/metabolism
- Sequence Alignment
- Sequence Homology, Amino Acid
- Tumor Suppressor Protein p53/chemistry*
- Tumor Suppressor Protein p53/genetics
- Tumor Suppressor Protein p53/metabolism
- Ubiquitin/chemistry*
- Ubiquitin/genetics
- Ubiquitin/metabolism
- Ubiquitin-Conjugating Enzymes/chemistry*
- Ubiquitin-Conjugating Enzymes/genetics
- Ubiquitin-Conjugating Enzymes/metabolism
- Ubiquitination
- Xenopus
- Zebrafish
- PubMed
- 33450248 Full text @ J. Mol. Biol.
Citation
Magnussen, H.M., Huang, D.T. (2021) Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant. Journal of molecular biology. 433:166807.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping