Characterizing the protein–protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization
Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization 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. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization 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 stabilizers.
Funding
University of Leicester
Irreversible Molecular Glues to Activate Anti-Cancer Interactome of 14-3-3sigma
Engineering and Physical Sciences Research Council
Find out more...A new mass spectrometer for structural proteomics and protein imaging
Biotechnology and Biological Sciences Research Council
Find out more...Midlands Integrative Biosciences Training Partnership (MIBTP)
Biotechnology and Biological Sciences Research Council
Find out more...University of Birmingham funded Midlands Integrative Biosciences Training Partnership (MIBTP2) (BB/M01116X/1)
The Eclipse mass spectrometer was funded by the BBSRC (BB/S019456/1)
History
Author affiliation
College of Life Sciences/Molecular & Cell BiologyVersion
- VoR (Version of Record)