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

Theintrinsically disordered 4E-BP2 protein regulates mRNA cap-dependenttranslation through interaction with the predominantly folded eukaryoticinitiation factor 4E (eIF4E). Phosphorylation of 4E-BP2 dramaticallyreduces the level of eIF4E binding, in part by stabilizing a binding-incompatiblefolded domain. Here, we used a Rosetta-based sampling algorithm optimizedfor IDRs to generate initial ensembles for two phospho forms of 4E-BP2,non- and 5-fold phosphorylated (NP and 5P, respectively), with the5P folded domain flanked by N- and C-terminal IDRs (N-IDR and C-IDR,respectively). We then applied an integrative Bayesian approach toobtain NP and 5P conformational ensembles that agree with experimentaldata from nuclear magnetic resonance, small-angle X-ray scattering,and single-molecule Fo''rster resonance energy transfer (smFRET).For the NP state, inter-residue distance scaling and 2D maps revealedthe role of charge segregation and pi interactions in driving contactsbetween distal regions of the chain (& SIM;70 residues apart). The5P ensemble shows prominent contacts of the N-IDR region with thetwo phosphosites in the folded domain, pT37 and pT46, and, to a lesserextent, delocalized interactions with the C-IDR region. Agglomerativehierarchical clustering led to partitioning of each of the two ensemblesinto four clusters with different global dimensions and contact maps.This helped delineate an NP cluster that, based on our smFRET data,is compatible with the eIF4E-bound state. 5P clusters were differentiatedby interactions of C-IDR with the folded domain and of the N-IDR withthe two phosphosites in the folded domain. Our study provides botha better visualization of fundamental structural poses of 4E-BP2 anda set of falsifiable insights on intrachain interactions that biasfolding and binding of this protein.

Automated summary

The intrinsically disordered protein 4E-BP2 interacts with eIF4E to regulate mRNA translation. Phosphorylation of 4E-BP2 reduces its binding to eIF4E. This study generated structural models of 4E-BP2 in different phosphorylation states and validated them using experimental data. The results provide insights into the interplay between different regions of 4E-BP2 and the effects of phosphorylation on these interactions.