title: Identification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometry.
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.
authors.affiliation: Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Hernychova
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.forename: Chandra
authors.initials: C
authors.lastname: Verma
authors.forename: Jude
authors.initials: J
authors.lastname: Nicholson
authors.forename: Carrie-Anne
authors.initials: CA
authors.lastname: Sharma
authors.forename: Eva
authors.initials: E
authors.lastname: Ruckova
authors.forename: Jin Yuan
authors.initials: JY
authors.lastname: Teo
authors.forename: Kathryn
authors.initials: K
authors.lastname: Ball
authors.forename: Borek
authors.initials: B
authors.lastname: Vojtesek
authors.forename: Ted R
authors.initials: TR
authors.lastname: Hupp
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement / Q000379:methods*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010879:Piperazines / Q000737:chemistry* / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D007093:Imidazoles; D010446:Peptide Fragments; D010879:Piperazines; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2
keywords: Allostery; Biomedicine; Cancer; Deuterium exchange; MDM2; p53