Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

Abstract

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin-proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or "lid" in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2(S17D)) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg(97)/Lys(98), and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.

The molecular dynamics of MDM2

“…The N-terminal domain is adjacent to an unstructured "lid" motif 2,3 that regulates E3 ligase function. 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.…”

Chemical states of the N-terminal “lid” of MDM2 regulate p53 binding: Simulations reveal complexities of modulation

“…Enzymological studies 12 have since shown that the phosphomimetic S17D can promote the binding of p53 to MDM2, and can increase the ubiquitination of p53 in vitro. These enzymological studies contrast to the predictions based on NMR and was rationalized through modeling which suggested that D17 forms a salt bridge (SB) with R97 and K98, both of which are located on the surface of MDM2 but away from the binding pocket.…”

CK1α Plays a Central Role in Mediating MDM2 Control of p53 and E2F-1 Protein Stability

“…MDM2 has been dissected into multiple functional domains: an N-terminal allosteric hydrophobic pocket that interacts with specific linear peptide docking motifs in proteins such as p53, a nuclear localization signal and a nuclear export signal, an acidic domain that binds the ubiquitin signal in the DNA-binding domain of p53, a C-terminal RING domain that coordinates E3 functions in ubiquitin transfer, an ATP-binding motif that regulates the chaperone functions of MDM2, and a pseudo-substrate motif or lid that regulates its ubiquitin ligase function (7). Reconstitution of the ubiquitin ligase function of MDM2 has demonstrated a two-site docking model for modification of p53.…”