Abstract
Upon phosphorylation of specific sites, eukaryotic translation initiation factor 4E (eIF4E) binding protein 2 (4E-BP2) undergoes a fundamental structural transformation from a disordered state to a four-stranded β-sheet, leading to decreased binding affinity for its partner. This change reflects the significant effects of phosphate groups on the underlying energy landscapes of proteins. In this study, we combine high-temperature molecular dynamics simulations and discrete path sampling to construct energy landscapes for a doubly phosphorylated 4E-BP2(18-62) and two mutants (a single site mutant D33K and a double mutant Y54A/L59A). The potential and free energy landscapes for these three systems are multifunneled with the folded state and several alternative states lying close in energy, suggesting perhaps a multifunneled and multifunctional protein. Hydrogen bonds between phosphate groups and other residues not only stabilize these low-lying conformations to different extents but also play an important role in interstate transitions. From the energy landscape perspective, our results explain some interesting experimental observations, including the low stability of doubly phosphorylated 4E-BP2 and its moderate binding to eIF4E and the inability of phosphorylated Y54A/L59A to fold.