Autoinhibition of MDMX by intramolecular p53 mimicry

Abstract

The p53 inhibitor MDMX is controlled by multiple stress signaling pathways. Using a proteolytic fragment release (PFR) assay, we detected an intramolecular interaction in MDMX that mechanistically mimics the interaction with p53, resulting in autoinhibition of MDMX. This mimicry is mediated by a hydrophobic peptide located in a long disordered central segment of MDMX that has sequence similarity to the p53 transactivation domain. NMR spectroscopy was used to show this hydrophobic peptide interacts with the N-terminal domain of MDMX in a structurally analogous manner to p53. Mutation of two critical tryptophan residues in the hydrophobic peptide disrupted the intramolecular interaction and increased p53 binding, providing further evidence for mechanistic mimicry. The PFR assay also revealed a second intramolecular interaction between the RING domain and central region that regulates MDMX nuclear import. These results establish the importance of intramolecular interactions in MDMX regulation, and validate a new assay for the study of intramolecular interactions in multidomain proteins with intrinsically disordered regions.T he p53 tumor suppressor is activated by numerous cellular and environmental signals and induces the expression of genes that regulate metabolism, cell growth, cell cycle, apoptosis, and senescence (1). MDM2 and MDMX are key regulators that control the cellular level and transcriptional activity of p53 through direct binding. Mouse knockout experiments showed that both MDM2 and MDMX are essential for controlling p53 activity during embryogenesis. Somatic knockout experiments showed that MDM2 is indispensable for regulating p53 in adult tissues, whereas MDMX deletion does not lead to cell death (2). MDM2 is a well-established p53 transcriptional target that forms a negative feedback loop by binding to the N-terminal transcriptional activation domain of p53 and, subsequently, ubiquitinating the C-terminal regulatory domain, which leads to degradation of p53 by the proteasome. p53 binding sites are also found in intron 1 of human MDMX, and p53 activation leads to moderate induction of MDMX transcription (3). Therefore, MDMX is a p53 target gene that may also provide dynamic feedback in response to p53 activation.MDMX alone does not have E3 ligase activity, but it is important for regulating p53 transcriptional function. MDMX expression and phosphorylation by the ATM/Chk2 pathway is important for the p53-mediated DNA damage response in mice (4, 5). MDMX levels are controlled by MDM2-mediated ubiquitination in a stress-dependent fashion (6, 7). Significant degradation of MDMX occurs after DNA damage through phosphorylation at several C-terminal sites (S342 and S367 by Chk2, S403 by ATM) (8). Furthermore, ribosomal stress promotes MDMX degradation through L11-MDM2 interaction (9), and oncogenic stress promotes MDMX degradation through ARF expression (10). Therefore, key signaling mechanisms that block p53 degradation simultaneously enhance MDMX degradation by MDM2. These observations underscore the co...

Functional Interrogation of the N-Terminal Lid of MDMX in p53 Binding <i>via</i> Native Chemical Ligation

“…4A). The K d value of (24-108) MDMX for the (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)p53 peptide is 200 nM, similar to the previously reported value of 119 nM measured by an FP-based direct binding study. 20) By contrast, (24-108)MDMX bound to (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) p53 at an affinity of 343 nM, representing a 1.7-fold decrease in p53 peptide binding affinity ascribe to the MDMX lid.…”

“…Apparently, the binding affinities of (1-108)MDMX for three p53 peptides with different length were slightly lower than that of (24- showed that MDMX contained an auto-inhibitory sequence which is encircled near residues Trp200 and Trp201, which competed intra-molecularly for binding with p53TAD. 27,28) The binding affinity of MDMX for p53 could be increased by remove or mutation of the sequence. Our results also suggest that the flexible lid of MDMX has rare auto-inhibitory function for p53 binding.…”

“…1 -Thr 108 was performed under similar conditions and the product (1-108)MDMX was purified and verified by preparative RP-HPLC and by ESI-MS. To achieve protein folding, 6 M Gu·HCl was used to dissolve the polypeptide at 1 mg/mL, while PBS (pH 7.4) was used for a six times dilution as well as a following extensive dialysis. For FP assays, (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)p53-TAMRA was achieved by covalently conjugate succinimidyl ester-activated carboxyfluorescein to the amino group of Lys24 in N-acetyl- (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)p53. Tecan Infinite M1000 was used to perform FP assays in 96-well plates.…”

“…Biacore T100 instrument was used to perform SPR-based direct binding assays at 25°C with a CM5 sensor chip and HBS-EP buffer. (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)p53 was immobilized to the sensor chip by its Lys24 side chain or N-terminus. Various concentrations of (1-108)MDMX (B) and (24-108) MDMX (C) proteins were injected onto the CM5 sensor chip.…”

Allosteric Interactions by p53 mRNA Govern HDM2 E3 Ubiquitin Ligase Specificity under Different Conditions

“…The E2-E3 pair could play a role in substrate specificity under different cellular conditions. Another question that arises from this work is the role of intramolecular interactions that has recently been described for HDMX (45,46) and for HDM2 (40). These intramolecular interactions could add another level of regulation to the way the domains are exposed to the solvent to allow highly accurate control of the interactome of HDM2.…”