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Understanding the interaction of 14-3-3 proteins with hDMX and hDM2: a structural and biophysical study
Sonja Srdanović1,2, Madita Wolter3,4, Chi H Trinh1,5
1Astbury Centre for Structural Molecular Biology, University of Leeds, UK.
Abstract:
p53 plays a critical role in regulating diverse biological processes: DNA repair, cell cycle arrest, apoptosis and senescence. The p53 pathway has therefore served as the focus of multiple drug-discovery efforts. p53 is negatively regulated by hDMX and hDM2; prior studies have identified 14-3-3 proteins as hDMX and hDM2 client proteins. 14-3-3 proteins are adaptor proteins that modulate localization, degradation and interactions of their targets in response to phosphorylation. Thus, 14-3-3 proteins may indirectly modulate the interaction between hDMX or hDM2 and p53 and represent potential targets for modulation of the p53 pathway. In this manuscript, we report on the biophysical and structural characterization of peptide/protein interactions that are representative of the interaction between 14-3-3 and hDMX or hDM2. The data establish that proximal phosphosites spaced ~20-25 residues apart in both hDMX and hDM2 co-operate to facilitate high-affinity 14-3-3 binding and provide structural insight that can be utilized in future stabilizer/inhibitor discovery efforts.
Insights
The tumor suppressor p53 pathway is modulated by 14-3-3 proteins binding to hDMX and hDM2. Proximal phosphosites on hDMX and hDM2 cooperate for high-affinity 14-3-3 binding, offering drug discovery targets.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- The p53 pathway is crucial for cellular functions including DNA repair, cell cycle arrest, apoptosis, and senescence.
- p53 activity is negatively regulated by hDMX and hDM2, making this pathway a key target for drug discovery.
- 14-3-3 proteins act as adaptor proteins, modulating the localization, degradation, and interactions of their targets, including hDMX and hDM2, upon phosphorylation.
Purpose of the Study:
- To characterize the biophysical and structural basis of the interaction between 14-3-3 proteins and peptides representing hDMX and hDM2.
- To understand how proximal phosphosites in hDMX and hDM2 contribute to 14-3-3 binding affinity.
- To provide structural insights for the development of novel stabilizers or inhibitors targeting the p53 pathway.
Main Methods:
- Biophysical characterization of protein-peptide interactions.
- Structural analysis of 14-3-3 binding interfaces.
- Site-directed mutagenesis to investigate the role of phosphosites.
Main Results:
- Demonstrated that proximal phosphosites, spaced approximately 20-25 residues apart in hDMX and hDM2, are essential for high-affinity 14-3-3 binding.
- Elucidated the structural mechanisms underlying cooperative binding of 14-3-3 proteins to these dual phosphosites.
- Identified key interaction interfaces that can be targeted for therapeutic intervention.
Conclusions:
- The cooperative binding of 14-3-3 proteins to dual phosphosites in hDMX and hDM2 is critical for regulating p53.
- Structural insights gained from this study can guide the rational design of small molecules to modulate the p53 pathway.
- Targeting the 14-3-3/hDMX/hDM2 interaction represents a promising strategy for cancer therapy.
