Edinburgh Napier University

  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 λmax 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 Asp17mutation inMDM2 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 fromE2 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 ofMDM2 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 theE2-ubiquitin thioester bond. These
data also suggest a new strategy to disrupt MDM2 function by targeting the E2-ubiquitin discharge reaction.