Systematic Mutational Analysis of Peptide Inhibition of the p53-MDM2/MDMX Interactions

Chong Li, Marzena Pazgier, Changqing Li, Weirong Yuan, Min Liu, Gang Wei, Wei Yue Lu, Wuyuan Lu*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

114 Scopus citations

Abstract

Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a potent duodecimal peptide inhibitor, termed PMI (TSFAEYWNLLSP), of the p53-MDM2 and -MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly 2 orders of magnitude higher than that of 17-28p53 (ETFSDLWKLLPE) of the same length; both peptides adopt nearly identical γ-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp, and Leu dominate PMI or 17-28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and 17-28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and 17-28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual-specific antagonist of MDM2 and MDMX reported to date, registering respective Kd values of 490 pM and 2.4 nM. The co-crystal structure of N8A-PMI-25-109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized intermolecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53-MDM2/MDMX interactions.

Original languageEnglish
Pages (from-to)200-213
Number of pages14
JournalJournal of Molecular Biology
Volume398
Issue number2
DOIs
StatePublished - Apr 2010
Externally publishedYes

Keywords

  • Alanine scanning mutagenesis
  • Crystallography
  • MDM2
  • MDMX
  • P53

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