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|Title:||Identification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometry||Authors:||Hernychova, Lenka
Teo, Jin Yuan
Hupp, Ted R.
|Keywords:||DRNTU::Science::Biological sciences||Issue Date:||2013||Source:||Hernychova, L., Man, P., Verma, C., Nicholson, J., Sharma, C. A., Ruckova, E., et al. (2013). Identification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometry. PROTEOMICS, 13(16), 2512-2525.||Series/Report no.:||PROTEOMICS||Abstract:||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 (MDM21–126) 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 MDM2Y104G and MDM2L107G mutants were fully active in p53 binding. However, the authentic p53-derived peptide:MDM2Y104G complex exhibited partial resistance to Nutlin-3 inhibition, while the p53-mimetic 12.1 peptide:MDM2Y104G 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.||URI:||https://hdl.handle.net/10356/100967
|ISSN:||1615-9853||DOI:||10.1002/pmic.201300029||Rights:||© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.||Fulltext Permission:||none||Fulltext Availability:||No Fulltext|
|Appears in Collections:||SBS Journal Articles|
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