Mdm2 and MdmX are important negative regulators of the tumor suppressor p53. Structurally homologous Mdm2 and MdmX inhibit p53 by directly blocking p53 transcriptional activation. Mdm2 also modifies and targets p53 for 265 proteasome dependent protein degradation through E3 ligase activity mediated by its C-terminal RING domain. However, MdmX lacks intrinsic E3 ligase activity and fails to catalyze ubiquitination of p53 despite containing a conserved RING domain. Thus, a comparative structural analysis between the Mdm2 and MdmX RING domains offers a unique way to elucidate the distinct functions of the two proteins in ubiquitination. We performed site-directed mutagenesis of the MdmX RING domain and found that the substitution of the residue N448 for cysteine and the substitution of the residue K478 for arginine granted MdmX RING domain ubiquitination activity. The structural analysis of the Mdm2 and MdmX RING domains revealed that the residue C449 of Mdm2 (structurally homologous to MdmX RING N448) located at the Mdm2 RING dimer interface is critical for the stability of the RING dimer structure, while the residue R479 (structurally homologous to MdmX RING K478) plays a role in recruiting and activating the ubiquitin E2 conjugating enzyme. This study provides new insight into the molecular mechanism of Mdm2 RING domain mediated ubiquitination. (C) 2014 Elsevier Inc. All rights reserved.