A novel p53 phosphorylation site within the MDM2 ubiquitination signal: II. a model in which phosphorylation at SER269 induces a mutant conformation to p53.

The p53 DNA-binding domain harbors a conformationally flexible multiprotein binding site that regulates p53 ubiquitination. A novel phosphorylation site exists within this region at Ser(269), whose phosphomimetic mutation inactivates p53. The phosphomimetic p53 (S269D) exhibits characteristics of mutant p53: stable binding to Hsp70 in vivo, elevated ubiquitination in vivo, inactivity in DNA binding and transcription, increased thermoinstability using thermal shift assays, and λ(max) of intrinsic tryptophan fluorescence at 403 nm rather than 346 nm, characteristic of wild type p53. These data indicate that p53 conformational stability is regulated by a phosphoacceptor site within an exposed flexible surface loop and that this can be destabilized by phosphorylation. To test whether other motifs within p53 have similarly evolved, we analyzed the effect of Ser(215) mutation on p53 function because Ser(215) is another inactivating phosphorylation site in the conformationally flexible PAb240 epitope. The p53(S215D) protein is inactive like p53(S269D), whereas p53(S215A) is as active as p53(S269A). However, the double mutant p53(S215A/S269A) was transcriptionally inactive and more thermally unstable than either individual Ser-Ala loop mutant. Molecular dynamics simulations suggest that (i) solvation of phospho-Ser(215) and phospho-Ser(269) by positive charged residues or solvent water leads to local unfolding, which is accompanied by local destabilization of the N-terminal loop and global destabilization of p53, and (ii) the double alanine 215/269 mutation disrupts hydrogen bonding normally stabilized by both Ser(215) and Ser(269). These data indicate that p53 has evolved two serine phosphoacceptor residues within conformationally flexible epitopes that normally stabilize the p53 DNA-binding domain but whose phosphorylation induces a mutant conformation to wild type p53.