Please use this identifier to cite or link to this item:
https://hdl.handle.net/10356/100967
Full metadata record
DC Field | Value | Language |
---|---|---|
dc.contributor.author | Hernychova, Lenka | en |
dc.contributor.author | Man, Petr | en |
dc.contributor.author | Verma, Chandra | en |
dc.contributor.author | Nicholson, Jude | en |
dc.contributor.author | Sharma, Carrie-Anne | en |
dc.contributor.author | Ruckova, Eva | en |
dc.contributor.author | Teo, Jin Yuan | en |
dc.contributor.author | Ball, Kathryn | en |
dc.contributor.author | Vojtesek, Borek | en |
dc.contributor.author | Hupp, Ted R. | en |
dc.date.accessioned | 2014-03-27T08:53:58Z | en |
dc.date.accessioned | 2019-12-06T20:31:32Z | - |
dc.date.available | 2014-03-27T08:53:58Z | en |
dc.date.available | 2019-12-06T20:31:32Z | - |
dc.date.copyright | 2013 | en |
dc.date.issued | 2013 | en |
dc.identifier.citation | 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. | en |
dc.identifier.issn | 1615-9853 | en |
dc.identifier.uri | https://hdl.handle.net/10356/100967 | - |
dc.description.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. | en |
dc.language.iso | en | en |
dc.relation.ispartofseries | PROTEOMICS | en |
dc.rights | © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. | en |
dc.subject | DRNTU::Science::Biological sciences | en |
dc.title | Identification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometry | en |
dc.type | Journal Article | en |
dc.contributor.school | School of Biological Sciences | en |
dc.identifier.doi | 10.1002/pmic.201300029 | en |
item.fulltext | No Fulltext | - |
item.grantfulltext | none | - |
Appears in Collections: | SBS Journal Articles |
SCOPUSTM
Citations
20
27
Updated on Mar 22, 2024
Web of ScienceTM
Citations
10
25
Updated on Oct 25, 2023
Page view(s) 50
597
Updated on Mar 28, 2024
Google ScholarTM
Check
Altmetric
Items in DR-NTU are protected by copyright, with all rights reserved, unless otherwise indicated.