Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX

Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2009; 106(12):4665-70. DOI: 10.1073/pnas.0900947106
Source: PubMed


The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53--a cellular process initiated by MDM2 and/or MDMX binding to the N-terminal transactivation domain of p53. MDM2 and MDMX in many tumors confer p53 inactivation and tumor survival, and are important molecular targets for anticancer therapy. We screened a duodecimal peptide phage library against site-specifically biotinylated p53-binding domains of human MDM2 and MDMX chemically synthesized via native chemical ligation, and identified several peptide inhibitors of the p53-MDM2/MDMX interactions. The most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 and MDMX at low nanomolar affinities--approximately 2 orders of magnitude stronger than the wild-type p53 peptide of the same length (ETFSDLWKLLPE). We solved the crystal structures of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6 A resolution. Comparative structural analysis identified an extensive, tightened intramolecular H-bonding network in bound PMI that contributed to its conformational stability, thus enhanced binding to the 2 oncogenic proteins. Importantly, the C-terminal residue Pro of PMI induced formation of a hydrophobic cleft in MDMX previously unseen in the structures of p53-bound MDM2 or MDMX. Our findings deciphered the structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX, shedding new light on structure-based rational design of different classes of p53 activators for potential therapeutic use.

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Available from: Guozhang Zou, Oct 05, 2015
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    • "We also examined the activities of several PMI-based p53- Mdm2 and p53-Mdm4 antagonists, as these are high-affinity Mdm2-binding peptides obtained through screening phage display libraries (Pazgier et al., 2009; Table S3; Supplemental Experimental Procedures). The lysate BiLC assay showed that all were highly potent p53-Mdm2 and p53-Mdm4 antagonists (Figure S6F). "
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    ABSTRACT: Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL's ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL's ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 11/2014; 9(5). DOI:10.1016/j.celrep.2014.10.058 · 8.36 Impact Factor
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    • "Hu et al. found a peptide (pDI) that binds to the p53-binding domain of MDM2 and inhibits the p53–MDM2 interaction34. Pazgier et al. determined the affinity of pDI towards MDM2 (Kd = 19.6 nM) and a p53-derived peptide (p53 pep) towards MDM2 (Kd = 140 nM)35. "
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    ABSTRACT: Detection of protein-protein interactions (PPIs) is important for understanding numerous processes in mammalian cells; however, existing PPI detection methods often give significant background signals. Here, we propose a novel PPI-detection method based on kinase-mediated growth induction of mammalian cells. In this method, target proteins are fused to the intracellular domain of c-kit (c-kit ICD) and expressed in interleukin-3-dependent mammalian cells. The PPI induces dimerization and activation of c-kit ICDs, which leads to cell growth in the absence of interleukin-3. Using this system, we successfully detected the ligand-dependent homo-interaction of FKBPF36V and hetero-interaction of FKBP and FRBT2098L, as well as the constitutive interaction between MDM2 and a known peptide inhibitor. Intriguingly, cells expressing high-affinity peptide chimeras are selected from the mixture of the cell populations dominantly expressing low-affinity peptide chimeras. These results indicate that this method can detect PPIs with low background levels and is suitable for peptide inhibitor screening.
    Scientific Reports 08/2014; 4:6127. DOI:10.1038/srep06127 · 5.58 Impact Factor
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    • "M06 binds into a compact and deep groove on the Mdm2 surface. It also positions the conserved p53-derived Mdm2 interaction motif (F19, W23, L26) into an identical orientation compared to the crystal structures elucidated for Mdm2 in complex with linear peptides either derived from the p53 WT sequence (PDB: 1YCR) [17] or the PMI variant selected for high affinity (PDB: 3EQS) [24]. Several key differences can however be observed when the Mdm2-M62A:M06 structure is overlaid with these structures. "
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    ABSTRACT: As key negative regulator of the p53 tumour suppressor, Mdm2 is an attractive therapeutic target. Small molecules such as Nutlin have been developed to antagonise Mdm2, resulting in p53-dependent death of tumour cells. We have recently described a mutation in Mdm2 (M62A), which precludes binding of Nutlin, but not p53. This Nutlin-resistant variant is not, however, refractory to binding and inhibition by stapled peptide antagonists targeting the same region of Mdm2. A detailed understanding of how stapled peptides are recalcitrant to Mdm2 mutations conferring Nutlin-resistance will aid in the further development of potent Mdm2 antagonists. Here, we report the 2.00 Å crystal structure of a stapled peptide antagonist bound to Nutlin resistant Mdm2. The stapled peptide relies on an extended network of interactions along the hydrophobic binding cleft of Mdm2 for high affinity binding. Additionally, as seen in other stapled peptide structures, the hydrocarbon staple itself contributes to binding through favourable interactions with Mdm2. The structure highlights the intrinsic plasticity present in both Mdm2 and the hydrocarbon staple moiety, and can be used to guide future iterations of both small molecules and stapled peptides for improved antagonists of Mdm2.
    PLoS ONE 08/2014; 9(8):e104914. DOI:10.1371/journal.pone.0104914 · 3.23 Impact Factor
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