The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo

Harumi Shimizu, Lindsay R Burch, Amanda J Smith, David Dornan, Maura Wallace, Kathryn L Ball, Ted R Hupp

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

Although the N-terminal BOX-I domain of the tumor suppressor protein p53 contains the primary docking site for MDM2, previous studies demonstrated that RNA stabilizes the MDM2.p53 complex using a p53 mutant lacking the BOX-I motif. In vitro assays measuring the specific activity of MDM2 in the ligand-free and RNA-bound state identified a novel MDM2 interaction site in the core domain of p53. As defined using phage-peptide display, the RNA.MDM2 isoform exhibited a notable switch in peptide binding specificity, with enhanced affinity for novel peptide sequences in either p53 or small nuclear ribonucleoprotein-U (snRNP-U) and substantially reduced affinity for the primary p53 binding site in the BOX-I domain. The consensus binding site for the RNA.MDM2 complex within p53 is SGXLLGESXF, which links the S9-S10 beta-sheets flanking the BOX-IV and BOX-V motifs in the core domain and which is a site of reversible conformational flexibility in p53. Mutation of conserved amino acids in the linker at Ser(261) and Leu(264), which bridges the S9-S10 beta-sheets, stimulated p53 activity from reporter templates and increased MDM2-dependent ubiquitination of p53. Furthermore, mutation of the conserved Phe(270) within the S10 beta-sheet resulted in a mutant p53, which binds more stably to RNA.MDM2 complexes in vitro and which is strikingly hyper-ubiquitinated in vivo. Introducing an Ala(19) mutation into the p53(F270A) protein abolished both RNA.MDM2 complex binding and hyper-ubiquitination in vivo, thus indicating that p53(F270A) protein hyper-ubiquitination depends upon MDM2 binding to its primary site in the BOX-I domain. Together, these data identify a novel MDM2 binding interface within the S9-S10 beta-sheet region of p53 that plays a regulatory role in modulating the rate of MDM2-dependent ubiquitination of p53 in cells.
Original languageEnglish
Pages (from-to)28446-58
Number of pages13
JournalJournal of Biological Chemistry
Issue number32
Publication statusPublished - 9 Aug 2002

Keywords / Materials (for Non-textual outputs)

  • Alleles
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Binding Sites
  • Dose-Response Relationship, Drug
  • Genes, p53
  • Humans
  • Immunohistochemistry
  • Leucine
  • Ligands
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Nuclear Proteins
  • Peptide Library
  • Precipitin Tests
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-mdm2
  • Sequence Homology, Amino Acid
  • Serine
  • Transcriptional Activation
  • Transfection
  • Tumor Cells, Cultured
  • Tumor Suppressor Protein p53
  • Ubiquitin


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