A Function for the RING Finger Domain in the Allosteric Control of MDM2 Conformation and Activity

Abstract

The MDM2 oncoprotein plays multiple regulatory roles in the control of p53-dependent gene expression. A picture of MDM2 is emerging where structurally discrete but interdependent functional domains are linked through changes in conformation. The domain structure includes: (i) a hydrophobic pocket at the N terminus of MDM2 that is involved in both its transrepressor and E3-ubiqutin ligase functions, (ii) a central acid domain that recognizes a ubiquitination signal in the core DNA binding domain of p53, and (iii) a C-terminal C2H2C4 RING finger domain that is required for E2 enzyme-binding and ATP-dependent molecular chaperone activity. Here we show that the binding affinity of MDM2s hydrophobic pocket can be regulated through the RING finger domain and that increases in pocket affinity are reflected by a gain in MDM2 transrepressor activity. Thus, mutations within the RING domain that affect zinc coordination, but not one that inhibits ATP binding, produce MDM2 proteins that have a higher affinity for the BOX-I transactivation domain of p53 and a reduced I 0.5 for p53 transrepression. An allosteric model for regulation of the hydrophobic pocket is supported by differences in protein conformation and pocket accessibility between wild-type and the RING domain mutant MDM2 proteins. Additionally the data demonstrate that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed toward its hydrophobic pocket.

Regulation of MDM2 E3 Ligase Activity by Phosphorylation after DNA Damage

“…These findings predict that the N-terminal p53 binding site and C-terminal RING domain may communicate directly or indirectly. In fact, a point mutation in the RING domain has been shown to change the conformation of the acidic domain and increase p53 binding by the N-terminal domain (47). There is also biochemical evidence that the RING domain of MDM2 binds to the acidic domain (11), suggesting the presence of an intramolecular interaction.…”

Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP

“…Interestingly, CHIP is not unique in acting as both a chaperone and an E3 ligase. The MDM2 E3 ligase is also known to possess an intrinsic chaperone activity that can be utilized to protect p53 from thermal denaturation in vitro and to promote folding of p53 to its native conformation in cells (32).…”

The molecular dynamics of MDM2

“…Such allostery is already foreshadowed; why do RING domain mutations increase intrinsic tryptophan fluorescence in the Acidic Domain and simultaneously increase Nutlin binding to the N-terminal hydrophobic pocket? 7 How does the "lid" of MDM2 interact with the gatekeeper residue Y100 to result in an "opening" or "closing" of the Acidic Domain of MDM2? Why is hydrophobic pocket occupation by Nutlin or p53 required for ubiquitination by MDM2?…”

“…4 The Acidic Domain is adjacent to a zinc-finger motif that regulates ribosomal stressresponses. 5 The RING domain forms a multi-functional dimer 6 containing an imbedded ATP binding motif that regulates the nucleolar localization and chaperone function of MDM2, 7,8 and an RNA-interaction site that regulates p53 translation. 9 Studies on the interaction between full-length MDM2 and p53 has revealed the importance of two dominant protein-protein interfaces giving rise to a dual-site model of MDM2-catalyzed ubiquitination.…”