Fig. 5.

Nutlin destabilizes the MDM2^S17D:p53 tetramer complex. a Strategy for measuring the stability of the MDM2:p53 complex in the presence of small ligands. Tetrameric forms of p53 protein were adsorbed onto the solid phase (in red) followed by MDM2 titration (gray) in the absence or presence of the indicated inhibitors, including (1) Nutlin-3, (2) BOX-Ia peptides, (3) BOX-Va or BOX-Vb peptides, and (4) DMSO carrier. The stability of MDM2 bound to p53 tetramers was measured using a monoclonal antibody specific for MDM2 by chemiluminescence. b and c MDM2^S17D binding to p53 is not inhibited by Nutlin. MDM2 (b) or MDM2^S17D (c) was assembled into reactions containing the indicated ligand: BOX-I peptide, BOX-V peptides, or Nutlin. The mixture was added to tetrameric p53 protein on the solid phase, and the amount of MDM2 bound stably to p53 was quantified using an MDM2 monoclonal antibody. The stability of the MDM2:p53 complex is depicted in relative light units