Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/100967
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dc.contributor.authorHernychova, Lenkaen
dc.contributor.authorMan, Petren
dc.contributor.authorVerma, Chandraen
dc.contributor.authorNicholson, Judeen
dc.contributor.authorSharma, Carrie-Anneen
dc.contributor.authorRuckova, Evaen
dc.contributor.authorTeo, Jin Yuanen
dc.contributor.authorBall, Kathrynen
dc.contributor.authorVojtesek, Boreken
dc.contributor.authorHupp, Ted R.en
dc.date.accessioned2014-03-27T08:53:58Zen
dc.date.accessioned2019-12-06T20:31:32Z-
dc.date.available2014-03-27T08:53:58Zen
dc.date.available2019-12-06T20:31:32Z-
dc.date.copyright2013en
dc.date.issued2013en
dc.identifier.citationHernychova, 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.en
dc.identifier.issn1615-9853en
dc.identifier.urihttps://hdl.handle.net/10356/100967-
dc.description.abstractMDM2 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.en
dc.language.isoenen
dc.relation.ispartofseriesPROTEOMICSen
dc.rights© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.en
dc.subjectDRNTU::Science::Biological sciencesen
dc.titleIdentification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometryen
dc.typeJournal Articleen
dc.contributor.schoolSchool of Biological Sciencesen
dc.identifier.doi10.1002/pmic.201300029en
item.fulltextNo Fulltext-
item.grantfulltextnone-
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