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*

Abstract

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 theBOX-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 β-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 Ser²⁶¹ and Leu²⁶⁴, which bridges the S9–S10 β-sheets, stimulated p53 activity from reporter templates and increased MDM2-dependent ubiquitination of p53. Furthermore, mutation of the conserved Phe²⁷⁰ within the S10 β-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 Ala¹⁹ mutation into the p53^F270A protein abolished both RNA·MDM2 complex binding and hyper-ubiquitinationin 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 β-sheet region of p53 that plays a regulatory role in modulating the rate of MDM2-dependent ubiquitination of p53 in cells.