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

Biophys J. 2022 Aug 16;121(16):3049-3060. doi: 10.1016/j.bpj.2022.07.015. Epub 2022 Jul 15.

Abstract

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamic characterization of their ensembles remain challenging, both in isolation and when they form dynamic "fuzzy" complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Förster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a non-uniform segmental flexibility around six different labeling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on nanosecond-to-microsecond timescales. Upon hyperphosphorylation, which induces folding of ∼40 residues in 4E-BP2, the quenching rates decreased at most labeling sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs significantly increased upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step toward a mechanistic understanding of this important IDP via integrative modeling.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Eukaryotic Initiation Factor-4E* / chemistry
  • Eukaryotic Initiation Factor-4E* / genetics
  • Eukaryotic Initiation Factor-4E* / metabolism
  • Eukaryotic Initiation Factors / chemistry
  • Eukaryotic Initiation Factors / genetics
  • Eukaryotic Initiation Factors / metabolism
  • Intrinsically Disordered Proteins* / chemistry
  • Phosphorylation
  • Protein Binding

Substances

  • Eukaryotic Initiation Factor-4E
  • Eukaryotic Initiation Factors
  • Intrinsically Disordered Proteins