Multisite phosphorylation and binding alter conformational dynamics of the 4E-BP2 protein

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Intrinsically disordered proteins (IDPs) are crucial for cellular regulation but challenging to characterize structurally.
• mRNA translation initiation involves eukaryotic initiation factor 4E (eIF4E) and intrinsically disordered 4E-binding proteins (4E-BPs) in a phosphorylation-dependent manner.

Purpose of the Study:

• To characterize the structural and dynamic ensemble of 4E-binding protein 2 (4E-BP2) in different states.
• To understand the dynamic interplay between 4E-BP2 and eIF4E during translation initiation regulation.

Main Methods:

• Single-molecule Förster resonance energy transfer (smFRET) to assess conformational changes.
• Fluorescence anisotropy decay and dynamic quenching with fluorescence correlation spectroscopy (FCS) to measure segmental flexibility and transient contacts.
• Time-resolved fluorescence techniques to map sequence-level rigidity.

Main Results:

• Binding to eIF4E induces non-uniform conformational changes in 4E-BP2, with a stiffer central region and increased dynamics in the C-terminal region.
• Phosphorylation of 4E-BP2 leads to partial folding and altered dynamics.
• Transient intra- and inter-molecular contacts occur on nanosecond-to-microsecond timescales.

Conclusions:

• The study provides a sequence-level rigidity map of 4E-BP2 in different states (bound/unbound, phosphorylated/unphosphorylated).
• Identifies specific regions of 4E-BP2 that interact with eIF4E and contribute to the complex's dynamics.
• Offers crucial insights for mechanistic understanding of translation regulation by IDPs.