Characterising the Protein-Protein Interaction Between MDM2 and 14-3-3σ; Proof of Concept for Small Molecule Stabilisation

Abstract

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumour suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of wild type p53. The 14-3-3 family of adaptor proteins are known to bind to MDM2 and the 14-3-3σ isoform controls MDM2 cellular localisation and stability to inhibit its activity. Therefore, small molecule stabilisation of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. In this work we provide a detailed biophysical and structural characterisation of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. Each of the two phosphorylated stretches of MDM2 occupies one of the two binding grooves of a 14-3-3σ dimer, a novel model for binding of di-phosphorylated peptides to 14-3-3 proteins. In addition, we show that the 14-3-3σ/MDM2 interaction is amenable to small molecule stabilisation. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in stablisation of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilisers.

Rapid Flow-Based Synthesis of Post-Translationally Modified Peptides and Proteins: A Case Study on MYC’s Transactivation Domain

“…The combination of nMS and NMR has recently been shown to be highly effective in examining the effect of PTMs and small molecules on PPIs. 50 In nMS, non-covalent interactions are maintained within protein complexes, 51 providing qualitative information on the extent of binding between Bin1 and various MYC peptides. Through this, we observed distinct effects on the MYC:Bin1 interaction depending on MYC's PTM-state and sequence-length.…”