Fig. 5: Importance of the structural features at the N terminus of the MDM2 RING domain homodimer.

a Cartoon representation of the structure of cat MDM2-422–C-S429E. b Cartoon representation of the MDM2-MDMX portion of MDM2-MDMX-UbcH5B–Ub complex structure (PDB ID: 5MNJ; left panel) and MDM2-MDMX RING domain structure (PDB ID: 2VJF; right panel) shown in the same view as in a. c Comparison of the location of pS429 in cat MDM2-422–C-pS429-UbcH5B–Ub structure and S429 in MDM2-MDMX-UbcH5B–Ub structure (PDB ID: 5MNJ). The MDM2-MDMX RING domain from the structure of MDM2-MDMX-UbcH5B–Ub complex was superimposed onto the structure of cat MDM2-422–C-pS429-UbcH5B–Ub complex. d Comparison of the location of pS429 in cat MDM2-422–C-pS429-UbcH5B–Ub structure and S429 in MDM2-MDMX RING domain structure (PDB ID: 2VJF). The MDM2-MDMX structure was superimposed onto the structure of cat MDM2-422–C-pS429-UbcH5B–Ub complex. e Close-up views of dimer interaction at the N terminus of MDM2 in the structures of cat MDM2-422–C-pS429-UbcH5B–Ub complex (left panel) and MDM2-MDMX RING domain (PDB ID: 2VJF; right panel). For a–e, all coloring is as described in Fig. 2, except MDM2^R in the heterodimer is colored gray. Key residues in c–e are shown in sticks and atoms are colored as in Fig. 4f. Distances between the Cα atoms of pS429 and S429 are indicated in the homodimer in c and in the heterodimer in d. f, h, j Reduced SDS-PAGE showing autoubiquitination reactions catalyzed by GST-MDM2-419–C variants using fluorescently labeled Ub and visualized with an Odyssey CLx Imaging System. Asterisks indicate non-reducible E1–Ub product. In f, Ala_ins indicates insertion of an alanine between residues 429 and 430 in MDM2. Uncropped gel images and InstantBlue-stained gels are shown in Supplementary Fig. 5f–h. g, i, k Plots showing the relative ubiquitination activity of MDM2 variants in f, h, j, respectively. Data are presented as mean value ± SD from three independent experiments (n = 3).