Identification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometry

Lenka Hernychova, Petr Man, Chandra Verma, Jude Nicholson, Carrie-Anne Sharma, Eva Ruckova, Jin Yuan Teo, Kathryn Ball, Borek Vojtesek, Ted R. Hupp*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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 (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.
Original languageEnglish
Pages (from-to)2512-2525
Number of pages14
JournalProteomics
Volume13
Issue number16
DOIs
Publication statusPublished - Aug 2013

Keywords

  • Allostery
  • Biomedicine
  • Cancer
  • Deuterium exchange
  • MDM2
  • p53
  • P53 TUMOR-SUPPRESSOR
  • BINDING-SITE
  • IN-VIVO
  • UBIQUITINATION
  • HDM2
  • SIMULATIONS
  • MODULATION
  • ACTIVATION
  • CHAPERONES
  • PEPTIDES

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