Phosphomimetic mutation of the N-terminal lid of MDM2 enhances the polyubiquitination of p53 through stimulation of E2-ubiquitin thioester hydrolysis

J Mol Biol. 2015 Apr 24;427(8):1728-47. doi: 10.1016/j.jmb.2014.12.011. Epub 2014 Dec 24.


Mouse double minute 2 (MDM2) has a phosphorylation site within a lid motif at Ser17 whose phosphomimetic mutation to Asp17 stimulates MDM2-mediated polyubiquitination of p53. MDM2 lid deletion, but not Asp17 mutation, induced a blue shift in the λ(max) of intrinsic fluorescence derived from residues in the central domain including Trp235, Trp303, Trp323, and Trp329. This indicates that the Asp17 mutation does not alter the conformation of MDM2 surrounding the tryptophan residues. In addition, Phe235 mutation enhanced MDM2 binding to p53 but did not stimulate its ubiquitination function, thus uncoupling increases in p53 binding from its E3 ubiquitin ligase function. However, the Asp17 mutation in MDM2 stimulated its discharge of the UBCH5a-ubiquitin thioester adduct (UBCH5a is a ubiquitin-conjugating enzyme E2D 1 UBC4/5 homolog yeast). This stimulation of ubiquitin discharge from E2 was independent of the p53 substrate. There are now four known effects of the Asp17 mutation on MDM2: (i) it alters the conformation of the isolated N-terminus as defined by NMR; (ii) it induces increased thermostability of the isolated N-terminal domain; (iii) it stimulates the allosteric interaction of MDM2 with the DNA-binding domain of p53; and (iv) it stimulates a novel protein-protein interaction with the E2-ubiquitin complex in the absence of substrate p53 that, in turn, increases hydrolysis of the E2-ubiquitin thioester bond. These data also suggest a new strategy to disrupt MDM2 function by targeting the E2-ubiquitin discharge reaction.

Keywords: MDM2; RING domain; allostery; linear motifs; ubiquitination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Cell Line, Tumor
  • Humans
  • Hydrolysis
  • Mice
  • Molecular Sequence Data
  • Point Mutation*
  • Polyubiquitin / metabolism*
  • Protein Conformation
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-mdm2 / chemistry
  • Proto-Oncogene Proteins c-mdm2 / genetics*
  • Proto-Oncogene Proteins c-mdm2 / metabolism*
  • Tumor Suppressor Protein p53 / chemistry
  • Tumor Suppressor Protein p53 / metabolism*
  • Ubiquitin / metabolism
  • Ubiquitin-Conjugating Enzymes / metabolism*
  • Ubiquitination


  • Tumor Suppressor Protein p53
  • Ubiquitin
  • Polyubiquitin
  • UBE2D1 protein, human
  • Ubiquitin-Conjugating Enzymes
  • Proto-Oncogene Proteins c-mdm2