Structural comparison of the Mdm2 and Pirh2 RING domains. A, schematic diagram showing the domain structure of Mdm2 and Pirh2 and the sequence of their RING domains. The cysteines and histidines involved in zinc coordination are labeled in red. B, ribbon representation of the Pirh2 RING, Mdm2 RING domain (Protein Data Bank code 2HDP), and overlay of the Pirh2 RING domain with the Mdm2 RING domain subunit A, the Pirh2 RING domain is shown in purple, and the Mdm2 RING domain is shown in cyan. The last seven residues of Mdm2 are shown in wheat. C, surface and electrostatic representation of the Pirh2 RING and Mdm2 RING domain subunit A. The orientation of the proteins in each case is the same as in B. The hydrophobic residues on the E2 binding surface of RING domains are indicated in yellow for Pirh2 and cyan for Mdm2. The figures were prepared with PyMOL (61) for the surface representation and Molmol (62) for electrostatic representation.

Pirh2 and Mdm2 favor different lysine linkage in p53 ubiquitylation and ubiquitylate different p53 lysine sites. A, schematic diagram showing the domain structure of p53 and the distribution of ubiquitylation target lysines of Pirh2 and Mdm2 within the p53 molecule. Lysine residues modified by Pirh2 and Mdm2 were identified by mass spectrometry of the p53 ubiquitin adducts produced by the in vitro ubiquitylation assay. The asterisks indicate the Ub sites reported in the literature (36, 37). B, the ubiquitin conjugation sites within the p53 DNA-binding and tetramerization (TET) domains detected by MS. In vitro reaction products were subjected to SDS-PAGE, and high molecular weight products were analyzed by LC-ESI-MS/MS. The numbers of spectra observed at the indicated sites are shown.

Mutagenesis studies of the Mdm2 RING domain. A, Pirh2, Mdm2, and MdmX RING domain sequences aligned by conserved cysteine and histidine residues. Secondary structure topology of the Pirh2 RING is shown above, whereas that of Mdm2 and MdmX are shown below. α-Helices are colored red, and β-sheets are blue. The mutated residues are indicated by the asterisks (Pirh2) and carets (Mdm2). B, schematic diagram of the Mdm2 RING domain dimer showing the mutated residues in stick form. The table summarizes the activities of the Mdm2 RING domain mutants in autoubiquitylation and p53 ubiquitylation. C, activity of the Mdm2 RING domain mutants in autoubiquitylation and p53 ubiquitylation was evaluated through an in vitro ubiquitylation assay in the presence of ATP, E1, E2 (UBE2D2), His-ubiquitin, and p53. Mdm2 autoubiquitylation was detected using an antibody against Mdm2 (SMP14), and the ubiquitylated p53 was detected using monoclonal antibody against p53 (PAB1801).

The chemical shift mapping of UBE2D2 upon binding to the Pirh2 RING domain. A, the regions with the greatest chemical shift changes induced upon binding to the Pirh2 RING domain are colored on the transparent surface representation of UBE2D2. The region of helix α1 is colored in red, Loop L1 is blue, and Loop L2 is orange. The catalytic Cys⁸⁵ is colored in green. B, composite chemical shift changes versus residue number for UBE2D2 upon binding to the Pirh2 RING domain. The values shown were calculated by using the equation, Δ_comp = (Δδ_HN² + (Δδ_N/5)²)½. The secondary structure elements of UBE2D2 are shown at the top with an arrow for β-strands and a rectangle for α-helices. C, surface representation of Pirh2 (pink) and Mdm2 (pale blue) ring domain, indicating the conserved E2 binding site (yellow) and the extended E2 binding surface of Mdm2 contributed by the positively charged region (blue) and the C-terminal residues from the adjacent subunit (magenta).

Comparison of E3 ligase activity of the Mdm2 and Pirh2 RING domains. A, autoubiquitylation of Pirh2 (lanes 1–6) and Mdm2 (lanes 7–11). GST-Pirh2 (E3) and His-Mdm2 (E3) were subjected to an in vitro ubiquitylation assay in the presence or absence of ATP, E1, E2 (UBE2D2), His-tagged ubiquitin, and p53, as indicated. After the reaction, the samples were resolved by SDS-PAGE and detected by Western blot. Pirh2 autoubiquitylation (left, lanes 1–6) was blotted with monoclonal antibody against GST. Autoubiquitylation of Mdm2 was evaluated using a specific antibody against Mdm2 (SMP14). B, p53 ubiquitylation by Pirh2 (lanes 1–6) and Mdm2 (lanes 7–11). Ubiquitylated p53 was detected using monoclonal antibody against p53 (PAB1801) in the reactions with increasing amounts of Pirh2 or Mdm2, as indicated. C, polyubiquitylation by Pirh2 and Mdm2. Left, total ubiquitylation catalyzed by Pirh2 and Mdm2 and the free ubiquitin in these reactions were subjected to fluorescence immunoblotting analysis and detected using a monoclonal antibody against ubiquitin and a Cy3-labeled mouse IgG. Right, the amount of free ubiquitin reported as an average fluorescence intensity of the free ubiquitin bands in the reactions of three repeated experiments quantified by a Typhoon imager.

Mutagenesis studies of the Pirh2 RING domain. A, schematic diagram of the Pirh2 RING domain showing the mutated residues in stick form. The table summarizes the activities of the Pirh2 RING domain mutants in mediating autoubiquitylation and ubiquitylation of p53. B, activity of the Pirh2 RING domain mutants in autoubiquitylation and p53 ubiquitylation was evaluated through an in vitro ubiquitylation assay in the presence of ATP, E1, E2 (UBE2D2), His-ubiquitin, Pirh2 mutants, and p53. Pirh2 autoubiquitylation was detected using an antibody against GST, and the ubiquitylated p53 was detected using monoclonal antibody against p53 (PAB1801). An asterisk indicates the GST-Pirh2 aggregates in the reaction, which are not ubiquitylated GST-Pirh2 products, as confirmed by the anti-ubiquitin immunoblot (not shown).

Mutagenesis studies of the Mdm2 RING last seven C-terminal residues. A, schematic diagram of the Mdm2 RING domain dimer showing the mutated residues in stick form. The table summarizes the activities of the Mdm2 RING C-terminal mutants in p53 ubiquitylation. B, activity of the His-tagged Mdm2 RING C-terminal mutants in autoubiquitylation and p53 ubiquitylation was evaluated through an in vitro ubiquitylation assay in the presence of ATP, E1, E2 (UBE2D2), His-ubiquitin, and p53. Mdm2 autoubiquitylation was detected using an antibody against Mdm2 (SMP14), and the ubiquitylated p53 was detected using monoclonal antibody against p53 (PAB1801). C, p53 ubiquitylation by Mdm2 homodimer, Mdm2/MdmX heterodimer, MdmX homodimer, Mdm2 ΔC, and Pirh2. The total concentration of Mdm2, MdmX, and Pirh2 was equivalent in each reaction mixture (0.5 μm). D, GST-pull-down analyses to assess the ability of Mdm2 C-terminal mutants to form a heterodimer with GST-MdmX. Mdm2 C-terminal mutants were detected with fluorescence immunoblotting using a monoclonal antibody against the His₆ tag, and GST-MdmX was detected with an antibody against GST. The ability of each Mdm2 mutant to interact with GST-MdmX was quantified as the normalized ratio of Mdm2 mutants versus MdmX using Mdm2 WT/MdmX as 1.