Multifunnel Energy Landscapes for Phosphorylated Translation Repressor 4E-BP2 and Its Mutants

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.

Investigation of Phosphorylation-Induced Folding of an Intrinsically Disordered Protein by Coarse-Grained Molecular Dynamics

“…Since the transitions from the unfolded to folded state and vice versa are the most interesting parts of the MD trajectories, we plotted, in addition, the structures of transition states illustrating the folding and unfolding pathways of the system. The spheres in the structures correspond to phosphorylated Threonine (pT) and Arginine (R) residues, which, according to previous studies, − can form salt bridges and play a crucial role in the stabilization of a tertiary structure.…”

“…To this end, molecular dynamics (MD) simulations can serve as a good means of studying the details of the regulation mechanism, in this case the folding mechanism of phosphorylated 4E-BP2. Recently, several computational studies were carried out to understand the folding mechanism induced by phosphorylation, − in which the mechanism of how two phosphorylated residues can fold the protein along with key residues playing an important role in stabilization was identified. Both canonical and replica-exchange molecular dynamics (REMD) simulations have been employed in these investigations.…”

“…In particular, the canonical MD simulations were performed at different temperatures and started from the NMR structure with PDB ID 2MX4, which enabled the authors to study the unfolding process of 4E-BP2. In order to examine the folding of 4E-BP2, the REMD simulations were carried out, in which the temperature-unfolded structures were used as initial structures. − Unfortunately, in some cases, REMD simulations failed to find the experimentally determined folded state …”

The Energy Landscape Perspective: Encoding Structure and Function for Biomolecules

“…To understand these effects we explored the energy landscapes of the doubly phosphorylated wild type (pWT) protein and two mutants, p(D33K) and p(Y54A/L59A) (Kang et al, 2020). All the potential and free energy landscapes exhibit multifunnel organisation, with four states competing with the folded conformation in pWT.…”

Recent development of atomistic force fields and simulations of intrinsically disordered proteins

“…好于ff99SB-ILDN [37] 和ff99SB-ILDN-NMR [38] 的模拟结 果(图2). 此外, RSFF系列力场在蛋白质结构优化 [39] 、 IDPs的模拟 [40,41] 和环状多肽的模拟 [42] 等方面也有很好 的表现.…”