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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    • "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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    • "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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    • "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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