Beyond disorder: An integrative investigation of the intrinsically disordered protein 4E-BP2.

Abstract

Intrinsically disordered proteins (IDPs) are prominent within the human proteome and play significant roles in neuronal processes. The 120-residue IDP 4E-BP2 (BP2) regulates translation initiation by interacting with eIF4E to form a complex, blocking eIF4G from attaching and preventing the start of translation. Hierarchical phosphorylation at 5 sites induces a disorder-to-order transition in which BP2 gains a four-stranded beta-fold domain spanning residues 18-62, drastically decreasing its binding affinity for eIF4E, thus regulating translation. Despite extensive efforts in characterizing the structural propensities of BP2, many questions remain regarding the structure-function relationship governing its role in the body. Experimental data from our labs including small-angle X-ray scattering, single molecule Förster resonance energy transfer (smFRET) and chemical shifts were integrated with Rosetta-based FastFloppyTail (Ferrie & Petersson, J. Phys. Chem. B, 2020) BP2 structures by means of the Bayesian-Maximum-Entropy (BME) algorithm (Bottaro et al, Methods Mol. Biol., 2020). BME balances confidence in the prior ensemble and reliability of experimental data using validation data in the form of paramagnetic relaxation enhancements. Solvent accessibility analysis and inter-residue distance maps of the optimized BP2 ensembles reveal increased accessibility in functionally relevant locations and surprising chain separations. Hierarchical clustering was performed by combining mathematically rigorous metrics and physically reasonable criteria. Cluster-level analysis of the conformational space revealed relevant structural features, including eIF4E-binding compatible structures of non-phosphorylated BP2, and contacts between the disordered N-terminus and folded domain for 5-phosphorylated BP2. Additionally, a sophisticated docking algorithm, AlphaFold Multimer (Evans et al, 2021), was applied to model the 4E-BP2:eIF4E complex. Dimensionality reduction techniques revealed the underlying low-dimensional space governing the motions of the disordered BP2 protein in complex with eIF4E, which were experimentally measured in a recent smFRET study from our labs (Smyth et al, Biophys. J., 2022).