Aggregation of the MDM2 RING domain is species dependent. (a) Superimposition of human MDM2 428-C (orange) and MDMX 428-C (dark-gray) (PDB: 5MNJ). Zinc ions are shown as gray spheres. The 3₁₀-helix preceding the RING domain is indicated. (b) Superimposition of the MDM2 RING domain homodimer (PDB: 6SQO; the two monomeric MDM2 RING domains are colored in orange and light orange) and human MDM2-MDMX RING domain heterodimer (PDB: 5MNJ; MDM2 is orange and MDMX is dark-gray). Zinc ions are shown as gray spheres. (c) Sequence alignment of the C-terminal region of MDM2 from different species. The regions corresponding to the 3₁₀-helix in MDM2^h and the RING domain are indicated. Sequence deviations from human MDM2 are highlighted in red. The sequence identity (id.) compared to human MDM2 is indicated on the right. (d, f, h) Superdex 75 elution profiles of MDM2^h, MDM2^f and MDM2^z, respectively (shown in black solid line). The elution profile of molecular weight markers (a: bovine serum albumin, 66 kDa; b: carbonic anhydrase, 29 kDa; c: cytochrome C, 12.4 kDa; d: aprotinin, 6,5 kDa) is shown as red dashed line. After removal of GST-tag, the cleaved MDM2 variants (expressed from 24L LB) were applied on a HiLoad 16/600 Superdex 75 column. (e, g, i) SDS-PAGE of indicated fractions from panels d, f, h, respectively. The position of the fractions within the elution profile is indicated by numbers (1–8).

Structural characterization of MDM2^f and MDM2^z. (a–c) Crystal structures of MDM2^z (a; purple/light purple), MDM2^f (b; blue/light blue) and MDM2^h (c; orange/light orange, PDB: 6SQO). Zinc ions are shown as gray spheres. The diameter of the dimer was calculated by measuring the distance between the Cα atoms of R471 (human nomenclature) of both protomers. (d) Superimposition of (a–c) in ribbon form. (e) Reduced SDS-PAGE showing autoubiquitination reactions catalyzed by GST-MDM2 variants using fluorescently-labeled Ub and visualized by an Odyssey CLx Imaging System (top panel) or stained with Coomassie Blue (bottom panel). (f) Crystal structure of the MDM2^f-UbcH5B–Ub complex. UbcH5B and Ub are colored in cyan and yellow, respectively. MDM2^f is colored as in b. (g,h) Close-up views of the key interactions between MDM2^f and UbcH5B involving MDM2^f’s I431 (g) and R470 (h),

SPR analyses of GST-MDM2 variants and UbcH5B–Ub binding affinities. Representative sensorgrams (left) and binding curves (right) for (a) MDM2^z and UbcH5B–Ub, (b) MDM2^f and UbcH5B–Ub, (c) MDM2^h and UbcH5B–Ub and (d) MDM2^hGT and UbcH5B–Ub. n = 2 for each binding curve.

Systematic analysis of MDM2 RING domain aggregation across species (a, c, e, g, i, k, m, o) Superdex 75 elution profiles of MDM2^h, MDM2^f, opossum MDM2 421-C, galago MDM2 425-C, MDM2^h A434S, MDM2^h I435V, MDM2^h G443T and MDM2^h P491S, respectively (shown as black solid line). The elution profile of molecular weight markers (a: bovine serum albumin, 66 kDa; b: carbonic anhydrase, 29 kDa; c: cytochrome C, 12.4 kDa; d: aprotinin, 6,5 kDa) is shown as red dashed line. GST-MDM2 variants (expressed from 2L LB) were treated with TEV then loaded on a Superdex 75 Increase 10/300 column. (b, d, f, h, j, l, n, p) SDS-PAGE showing the GST-MDM2 variants before and after TEV treatment to release the GST-tag (labeled ‘–’ and ‘+’, respectively) and single fractions of the corresponding SEC experiments from panels a, c, e, g, i, k, m, o, respectively. The position of the fractions within the elution profile is indicated by numbers (1–5).

Functional and structural characterization of MDM2^hGT. (a) Superdex 75 elution profile of MDM2^hGT from a large-scale purification (shown as black solid line). The elution profile of molecular weight markers (a: bovine serum albumin, 66 kDa;b: carbonic anhydrase, 29 kDa; c: cytochrome C, 12.4 kDa; d: aprotinin, 6,5 kDa) is shown as red dashed line. (b) SDS-PAGE showing the purity of single fractions from a. The position of the fractions within the elution profile is indicated by numbers (1–13). The large absorbance in fraction 1 is due to the presence of other contaminants. (c) Crystal structure of the MDM2^hGT-UbcH5B–Ub complex. The two MDM2^hGT monomers are colored in orange and light orange. Zinc ions are shown as gray spheres. UbcH5B and Ub are colored in cyan and yellow, respectively. A representative close-up view of the local environment of G443T including the sidechain of UbcH5B’s K4 are shown. (d,e) Reduced SDS-PAGE showing autoubiquitination reactions catalyzed by GST-MDM2^h and GST-MDM2^hGT using fluorescently-labeled Ub and visualized by an Odyssey CLx Imaging System (d) or stained with Coomassie Blue (e). Asterisk in d indicates non-reducible E1–Ub product.

3₁₀-helix precedes the RING domain in MDM2. (a) Close-up view of the 3₁₀-helix that precedes the RING domain in MDM2^h. Location of A434 within MDM2^h is indicated. (b) Close-up view of the corresponding region in MDM2^f. No electron density was observed in this region.