Abstract
Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.
Highlights
Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates capdependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex
We have previously demonstrated that the phosphorylation of T37 and T46 in apo 4E-BP2 induces folding of residues P18–R62 into a four β-stranded folded domain that partially sequesters the canonical eIF4E-binding helix, with pT37 and pT46 being part of conserved pTPGGT motifs that form hairpin turns central to the structure[11]
We found that hairpin turn propensity correlates with three markers related to ubiquitination (Fig. 8d–i), including (i) the presence of local lysine residues (Fig. 8d, g), (ii) the likelihood of matching one of the aforementioned phosphodegron motifs (Fig. 8e, h) and (iii) the likelihood of ubiquitination being directly observed in the local sequence (Fig. 8f, i)
Summary
Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates capdependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E bindingincompatible conformation and regulating translation initiation. Since dysregulation of eIF4E function is involved in many diseases including cancer and autism spectrum disorders[1,2] and ubiquitin-mediated degradation of 4E-BPs is dependent on its phosphorylation status[14], understanding the stepped binding affinities and the link with protein stability is critical. A low-resolution model based on SAXS data exists[23], no atomic structure of eIF4E in complex with any full-length 4E-
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