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

Authors

  • Lenka Hernychova,

    1. Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
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  • Petr Man,

    1. Academy of Sciences of the Czech Republic v.v.i, Institute of Microbiology, Czech Republic
    2. Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, Czech Republic
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  • Chandra Verma,

    1. Bioinformatics Institute (A*STAR), Singapore
    2. Department of Biological Sciences, National University of Singapore, Singapore
    3. School of Biological Sciences, Nanyang Technological University, Singapore
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  • Jude Nicholson,

    1. Cell Signalling Unit, p53 Signal Transduction Laboratories, Institute of Genetics and Molecular Medicine, University of Edinburgh Cancer Research Centre, Edinburgh, UK
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  • Carrie-Anne Sharma,

    1. Cell Signalling Unit, p53 Signal Transduction Laboratories, Institute of Genetics and Molecular Medicine, University of Edinburgh Cancer Research Centre, Edinburgh, UK
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  • Eva Ruckova,

    1. Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
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  • Jin Yuan Teo,

    1. Bioinformatics Institute (A*STAR), Singapore
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  • Kathryn Ball,

    1. Cell Signalling Unit, p53 Signal Transduction Laboratories, Institute of Genetics and Molecular Medicine, University of Edinburgh Cancer Research Centre, Edinburgh, UK
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  • Borek Vojtesek,

    1. Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
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  • Ted R. Hupp

    Corresponding author
    1. Cell Signalling Unit, p53 Signal Transduction Laboratories, Institute of Genetics and Molecular Medicine, University of Edinburgh Cancer Research Centre, Edinburgh, UK
    • Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
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  • Colour Online: See the article online to view Figs. 1, 3, and 5–10 in colour.

Correspondence: Professor Ted R. Hupp, Institute of Genetics and Molecular Medicine, University of Edinburgh Cancer Research Centre, CRUK p53 Signal Transduction Group, Crewe Road South, Edinburgh EH4 2XR, UK

E-mail: ted.hupp@ed.ac.uk

Fax: +441317773583

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.

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