Document Detail

Identification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometry.
MedLine Citation:
PMID:  23776060     Owner:  NLM     Status:  MEDLINE    
MDM2 is a multidomain protein that functions as an E3 ubiquitin ligase, transcription repressor, mRNA-binding protein, translation factor, and molecular chaperone. The small molecule Nutlin-3 has been engineered to bind to the N-terminal hydrophobic pocket domain of MDM2. This binding of Nutlin-3 has two consequences: (i) antagonistic effects through competitive disruption of the MDM2-p53 complex and (ii) agonist effects that allosterically stabilize MDM2 protein-protein interactions that increase p53 ubiquitination as well as nucleophosmin deoligomerization. We present a methodology using a hydrogen/deuterium (H/D) exchange platform that measures Nutlin-3 binding to the N-terminal domain of MDM2 (MDM2(1-126)) in order to begin to develop dynamic assays that evaluate MDM2 allostery. In order to localize the regions in MDM2 being suppressed by Nutlin-3, MDM2 was incubated with the ligand and H/D amide exchange was measured after pepsin digestion. One dynamic segment containing amino acids 55-60 exhibited slower deuterium exchange after Nutlin-3 binding, reflecting ligand binding within the hydrophobic pocket. However, another dominant suppression of H/D exchange was observed in a motif from amino acids 103-107 that reflects surface hydrophobic residues surrounding the hydrophobic pocket of MDM2. In order to explore the consequences of this latter Nutlin-3 interaction site on MDM2, the Y104G and L107G mutant series was constructed. The MDM2(Y104G) and MDM2(L107G) mutants were fully active in p53 binding. However, the authentic p53-derived peptide:MDM2(Y104G) complex exhibited partial resistance to Nutlin-3 inhibition, while the p53-mimetic 12.1 peptide:MDM2(Y104G) complex retained normal Nutlin-3 responsiveness. These data reveal the existence of a second functional Nutlin-3-binding site in a surface hydrophobic patch of MDM2, flanking the hydrophobic pocket. This reveals two modes of peptide binding by MDM2 and highlights the utility of H/D exchange as an assay for measuring allosteric effects in MDM2.
Lenka Hernychova; Petr Man; Chandra Verma; Jude Nicholson; Carrie-Anne Sharma; Eva Ruckova; Jin Yuan Teo; Kathryn Ball; Borek Vojtesek; Ted R Hupp
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Proteomics     Volume:  13     ISSN:  1615-9861     ISO Abbreviation:  Proteomics     Publication Date:  2013 Aug 
Date Detail:
Created Date:  2013-08-09     Completed Date:  2014-02-28     Revised Date:  2014-03-19    
Medline Journal Info:
Nlm Unique ID:  101092707     Medline TA:  Proteomics     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  2512-25     Citation Subset:  IM    
Copyright Information:
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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MeSH Terms
Allosteric Site
Amino Acid Sequence
Deuterium Exchange Measurement / methods*
Imidazoles / chemistry*,  metabolism
Models, Molecular
Molecular Sequence Data
Peptide Fragments
Piperazines / chemistry*,  metabolism
Proto-Oncogene Proteins c-mdm2 / chemistry*,  metabolism
Grant Support
11649//Cancer Research UK; //Biotechnology and Biological Sciences Research Council
Reg. No./Substance:
0/Imidazoles; 0/Peptide Fragments; 0/Piperazines; 0/nutlin 3; EC protein, human; EC Proteins c-mdm2

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