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Updated: Aug 12, 2025
Identifying Protein-protein Interaction Sites Using Peptide Arrays
Published on: November 18, 2014
Identification of peptide binding sequence of TRIM25 on 14-3-3σ by bioinformatics and biophysical techniques
De Chen Chiang1, Aik-Hong Teh2, Beow Keat Yap1
1School of Pharmaceutical Sciences, Universiti Sains Malaysia, USM, Penang, Malaysia.
Abstract:
14-3-3σ protein is one of the seven isoforms from the highly conserved eukaryotic 14-3-3 protein family. Downregulation of 14-3-3σ expression has been observed in various tumors. TRIM25 is responsible for the proteolytic degradation of 14-3-3σ, in which abrogation of TRIM25 suppressed tumor growth through 14-3-3σ upregulation. However, to date, the exact 14-3-3σ interacting residues of TRIM25 have yet to be resolved. Thus, this study attempts to identify the peptide binding sequence of TRIM25 on 14-3-3σ via both bioinformatics and biophysical techniques. Multiple sequence alignment of the CC domain of TRIM25 revealed five potential peptide binding sequences (Peptide 1-5). Nuclear magnetic resonance (NMR) assay (1H CPMG) identified Peptide 1 as an important sequence for binding to 14-3-3σ. Competition NMR assay suggested that Peptide 1 binds to the amphipathic pocket of 14-3-3σ with an estimated KD of 116.4 µM by isothermal titration calorimetry. Further in silico docking and molecular dynamics simulations studies proposed that Peptide 1 is likely to interact with Lys49, Arg56, Arg129, and Tyr130 residues at the amphipathic pocket of 14-3-3σ. These results suggest that Peptide 1 may serve as a biological probe or a template to design inhibitors of TRIM25-14-3-3σ interaction as a potentially novel class of anticancer agents.Communicated by Ramaswamy H. Sarma.
Insights
Researchers identified a specific peptide sequence (Peptide 1) on TRIM25 that binds to 14-3-3σ protein. This finding is crucial for developing new anticancer agents targeting tumor growth.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- 14-3-3σ protein expression is often reduced in tumors.
- TRIM25 degrades 14-3-3σ, and inhibiting TRIM25 can suppress tumor growth.
- The precise interaction sites between TRIM25 and 14-3-3σ are not fully understood.
Purpose of the Study:
- To identify the specific peptide binding sequence of TRIM25 responsible for interacting with 14-3-3σ.
- To characterize the binding interaction using biophysical and computational methods.
Main Methods:
- Bioinformatics analysis including multiple sequence alignment of TRIM25's CC domain.
- Biophysical techniques such as Nuclear Magnetic Resonance (NMR) and isothermal titration calorimetry (ITC).
- In silico molecular docking and molecular dynamics simulations.
Main Results:
- Five potential peptide binding sequences were identified in TRIM25's CC domain.
- NMR and ITC assays confirmed Peptide 1 as a key binding sequence for 14-3-3σ, with a KD of 116.4 µM.
- Computational studies indicated Peptide 1 interacts with specific residues (Lys49, Arg56, Arg129, Tyr130) in the 14-3-3σ amphipathic pocket.
Conclusions:
- Peptide 1 of TRIM25 is a critical interaction site with 14-3-3σ.
- This identified interaction can be targeted for developing novel anticancer therapies.
- Peptide 1 serves as a potential biological probe or a template for designing TRIM25-14-3-3σ interaction inhibitors.
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