Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant

Autor: Danny T. Huang, Helge M. Magnussen
Rok vydání: 2020
Předmět:
Models
Molecular
Protein Conformation
alpha-Helical
Xenopus
Regulator
Gene Expression
medicine.disease_cause
Crystallography
X-Ray
RMSD
Root-mean-square deviation
0302 clinical medicine
E3
Structural Biology
GST
Glutathione S-transferase
Catalytic Domain
Conserved Sequence
Zebrafish
Mammals
0303 health sciences
Mutation
biology
Chemistry
Proto-Oncogene Proteins c-mdm2
Small molecule
Recombinant Proteins
Ubiquitin ligase
MDM2h
human MDM2 419-C
Domain (ring theory)
TEV
Tobacco Etch Virus
Protein Binding
Research Article
SPR
Surface plasmon resonance
E2(UbcH5B)~Ub
thioester bond-linked E2~Ub conjugate
Protein design
E2(UbcH5B)–Ub
isopeptide-linked E2–Ub conjugate
Computational biology
Ring (chemistry)
03 medical and health sciences
Protein Aggregates
Aggregation
Protein Domains
MDM2
medicine
Animals
Humans
Protein Interaction Domains and Motifs
Amino Acid Sequence
Molecular Biology
neoplasms
030304 developmental biology
ComputingMethodologies_COMPUTERGRAPHICS
Sequence Homology
Amino Acid
Ubiquitin
Mutagenesis
Ubiquitination
MDM2z
Danio rerio MDM2 407-C
Ubiquitin-Conjugating Enzymes
biology.protein
Biocatalysis
Protein Conformation
beta-Strand
MDM2f
Xenopus tropicalis MDM2 414-C
Tumor Suppressor Protein p53
Protein Processing
Post-Translational
Sequence Alignment
030217 neurology & neurosurgery
Zdroj: Journal of Molecular Biology
ISSN: 1089-8638
0022-2836
Popis: Graphical abstract
Highlights • The MDM2 RING domain has a tendency to form aggregates, which is species dependent. • The structures of dimeric MDM2 RING domains from frog and fish are presented. • A G443T substitution strongly reduces aggregation of the human MDM2 RING domain. • MDM2-G443T exhibits similar structure and activity as wild-type MDM2.
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.
Databáze: OpenAIRE
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