Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 105651 |
| Journal | The Journal of biological chemistry |
| Volume | 300 |
| Issue number | 2 |
| Early online date | 16 Jan 2024 |
| DOIs | |
| Publication status | Published - Feb 2024 |
Bibliographical note
The authors would like to thank Dr Louise Fairall for assistance with the X-ray diffraction data collection, scaling, and analysis and Dr Sharad Mistry for mass spectrometry analysis of intact protein samples. The biophysical and structural research was funded by the University of Leicester and the Engineering and Physical Sciences Research Council (EP/W015803/1). The Eclipse mass spectrometer was funded by the BBSRC (BB/S019456/1). The mass spectrometry research was supported by the Biotechnology and Biological Sciences Research Council (BBSRC, BB/M01116X/1) and 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).P. R. was the recipient of a Research Project Prize 2021 (Grant code Plant EDEM) by the Department of Biological and Agro-alimentary Sciences of the Italian National Research Council.
