Delineating Structural Propensities of the 4E-BP2 Protein via Integrative Modeling and Clustering

Area of Science:

• Molecular Biology
• Structural Biology
• Biophysics

Background:

• The 4E-BP2 protein is intrinsically disordered and regulates mRNA translation by interacting with eukaryotic initiation factor 4E (eIF4E).
• Phosphorylation of 4E-BP2 alters its structure, reducing binding affinity to eIF4E by stabilizing a folded domain incompatible with binding.

Purpose of the Study:

• To generate and analyze conformational ensembles for non-phosphorylated (NP) and 5-fold phosphorylated (5P) 4E-BP2.
• To elucidate the role of intrachain interactions in the folding and binding dynamics of 4E-BP2.

Main Methods:

• Utilized a Rosetta-based sampling algorithm optimized for intrinsically disordered regions (IDRs).
• Employed an integrative Bayesian approach combined with experimental data (NMR, SAXS, smFRET).
• Applied agglomerative hierarchical clustering to analyze conformational ensembles.

Main Results:

• The NP state shows contacts driven by charge segregation and pi interactions between distant regions.
• The 5P state exhibits distinct interactions, with the N-terminal IDR (N-IDR) contacting phosphosites (pT37, pT46) in the folded domain.
• Identified an NP cluster compatible with eIF4E binding and differentiated 5P clusters based on C-terminal IDR (C-IDR) and N-IDR interactions.

Conclusions:

• Provided detailed structural insights into the conformational ensembles of NP and 5P 4E-BP2.
• Revealed how intrachain interactions and phosphorylation bias protein folding and eIF4E binding.
• Generated falsifiable hypotheses regarding the structural mechanisms of translation regulation by 4E-BP2.