Single-Molecule Dissection of the Conformations, Dynamics and Binding of the Disordered 4E-BP2 Protein

Abstract

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions. Cap-dependent translation initiation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with disordered eIF4E binding proteins (4E-BPs) in a phosphorylation dependent manner. Single molecule fluorescence resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), time-resolved fluorescence anisotropy (TRFA) and nuclear magnetic resonance (NMR) were used to detect and assess the structural changes and sequence-specific local chain motions of 4E-BP2 upon phosphorylation and upon binding to eIF4E.1H - 15N heteronuclear single quantum correlation (HSQC) spectra demonstrate that cysteine mutations of 4E-BP2 do not perturb its phosphorylated folded structure. Multiparameter smFRET analysis reveals changes in the conformational ensemble upon phosphorylation, denaturation and binding to elF4E. Nanosecond scale dynamics in 4E-BP2 were observed by site-specific FCS, and were tentatively assigned to formation of transient intrachain contacts. Our data suggests that multi-site phosphorylation of the protein slows down the proximal chain motions and also modulates the kinetics of distal regions. Segmental rotational correlation times and wobbling cone angles extracted for different sites along the chain provide a rigidity map of this IDP and can be used to evaluate its binding mode to eIF4E.