FC0072
Eukaryotic translation initiation factor 4E -binding protein 2 (4E-BP2)  -  Eukaryotic translation initiation factor 4E (eIF4E)

Biological function
Cap-dependent translation initiation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with eIF4E binding proteins (4E-BPs). The 4E-BPs play essential roles in development and cell growth in all cell types and, in neurons, 4E-BP2 is critical for synaptic plasticity, learning and memory formation.

Structural evidence
4E-BP2 possesses significant transient secondary structure throughout the protein with a high helical propensity in the canonical binding site. Five regions, M1-A5, D33-T37, T50-N64, T86-E89, and N96-D105, have helical propensities greater than 0.15. 4E-BP2 binds to eIF4E using a bipartite mode of interaction involving residues spanning from immediately N-terminal to the canonical binding site to beyond the highly conserved ⁷⁸IPGVT⁸² sequence. Interestingly, mutating ⁷⁸IPGVT⁸², but not ⁵⁴YXXXXLΦ⁶⁰, significantly changes the dynamics of the complex such that some previously invisible resonances of the bound state can be observed. Significant resonance broadening on the surface of eIF4E and chemical shift changes throughout eIF4E, demonstrating extensive allosteric effects of 4E-BP binding. The loss of bound resonances within the interaction interface likely originates from broadening due to microsecond to millisecond conformational exchange within the complex. In the complex with wild- type 4E- BP2, the secondary binding site is more dynamic than the canonical site. The regions of 4E-BP2 outside the binding interface maintain their intrinsically disordered nature, with ’flanking fuzziness’ and T19-A28 even more flexible in the bound state.

Biochemical evidence
Out of the three 4E-BP isoforms 4E-BP2 exhibits the highest affinity for eIF4E. 4E-BP2 contains significant fluctuating secondary structure and binds eIF4E at an extensive bipartite interface including the canonical ⁵⁴YXXXXLΦ⁶⁰ and ⁷⁸IPGVT⁸² sites. Each of the two binding elements individually has submicromolar affinity and exchange on and off of the eIF4E surface within the context of the overall nanomolar complex. Systematic deletion mutagenesis and surface plasmon resonance to demonstrate that the residues C-terminal to the canonical binding site contribute significantly to binding with the affinity of full-length 4E-BPs being approximately two to three orders of magnitude higher than those of the peptides. The KD value for eIF4E binding to wild-type 4E-BP2 is 3.2 ± 0.6 nM. The ⁷⁸IPGVT⁸² segment undergoes more conformational fluctuations within the bound state than the canonical site. Interactions involving both sites are synergistic, with a low nanomolar KD, since isolated canonical site peptides bind in the micromolar range. The binding of the canonical site is enthalpically driven, whereas interaction of the secondary binding site is driven by entropy, suggesting that this second site is highly dynamic in the complex. The dynamic nature of 4E-BP2 was observed in complexes with both cap-free and cap-bound eIF4E.

Structure/Mechanism
The striking differences between the NMR spectra of complexes with peptides and with full-length 4E-BPs suggest that a dynamic/fuzzy complex is formed, resulting in an ensemble of conformations being sampled in the bound state. The dynamic interactions facilitates exposure of regulatory phosphorylation sites within the complex. It is important to note that the N-terminal part of eIF4E also remains to be disordered in the complex.

Mechanism category
Tethering, Flexibility modulation

Posttranslational modification
Unphosphorylated or minimally phosphorylated 4E-BPs associate tightly with eIF4E to inhibit cap-dependent translation, whereas highly phosphorylated 4E-BPs dissociate from eIF4E, allowing translation to proceed. The tight interaction of the eIF4E:4E-BPs complex, low k_off rates (~1.2–3.7 3 10^_4/s) of 4E-BP2 and the fact that 4E-BP2 is in excess in vivo, 4E-BP2 phosphorylation likely occurs on the complex and phosphorylation sites must be accessible to large kinases in the bound state in order to enable the rapid biological response. Understanding the molecular mechanism of eIF4E and 4E-BP interaction is crucial because inhibiting 4E-BP phosphorylation or designing 4E-BP mimics may be useful in suppressing tumors.

Isoforms, context-dependence
The SPR analyses of the eIF4E–4E-BP pairs showed the binding preference in the order of 4E-BP2 > 4E-BP3 > 4E- BP1 regardless of the cap-free or cap-bound state of eIF4E. This preferred binding is mainly due to the differences in their dissociation rates, because no notable differences were observed among the k_a values of 4E-BP1–3. The participation of the N-terminal flexible region of eIF4E in the interactions with 4E-BP1 and 2 was clearly shown by the present SPR analysis. The deletion of 33 N-terminal residues of eIF4E markedly increased the binding affinities with 4E-BP1 and 4E- BP2 through the decreased kD values, whereas such a change was not observed by the N-terminal deletion up to 26 residues. This obviously indicates the suppressive or regulative function of N-terminal region, at least 1–26 sequence, of eIF4E for the interaction with 4E-BP1 and 2; the importance of the N-terminal region of eIF4E in its association with eIF4G, that shares the eIF4E-binding region with 4E-BP isoform, has been reported In contrast, the binding affinity with 4E-BP3 was hardly affected by the N-terminal deletion up to 33 residues, indicating no direct participation of N-terminal flexible region of eIF4E in the binding with 4E-BP3. From the comparison among the amino acid sequences of 4E-BP1–3, this could suggest that the N-terminal region of 4E-BP1 and 2 also participates importantly in the regulated binding to eIF4E, because the length of N-terminal region of 4E-BP3 is much shorter than those of 4E-BP1 and 4E-BP2.

Significance
Fuzziness enables long-range allosteric effects, which causes allosteric enhancement of eIF4E mRNA cap binding. Fuzziness also facilitates the regulation of translation initiation through posttranslational modifications such as phosphorylation by high frequency exposure of target sites to kinases and other enzymes.

Medical relevance
The 4E-BP2 interface on eIF4E overlaps yet is more extensive than the eIF4G:eIF4E interface, suggesting that these key interactions may be differentially targeted for therapeutics.

Further reading
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