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

Proteomics. 2013 Aug;13(16):2512-25. doi: 10.1002/pmic.201300029.


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.

Keywords: Allostery; Biomedicine; Cancer; Deuterium exchange; MDM2; p53.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Site
  • Amino Acid Sequence
  • Deuterium Exchange Measurement / methods*
  • Humans
  • Imidazoles / chemistry*
  • Imidazoles / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments
  • Piperazines / chemistry*
  • Piperazines / metabolism
  • Proto-Oncogene Proteins c-mdm2 / chemistry*
  • Proto-Oncogene Proteins c-mdm2 / metabolism


  • Imidazoles
  • Peptide Fragments
  • Piperazines
  • nutlin 3
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2