Importance of C-terminal flexible region of 4E-binding protein in binding with eukaryotic initiation factor 4E.

Although the alpha-helical Y(X)4Lvarphi containing region of eIF4E-binding protein (4EBP) is the major binding region with eukaryotic initiation factor 4E (eIF4E), the roles of its N- and C-terminal regions in the binding are hardly known. To clarify the roles of these flexible regions in the interaction, the binding features of the sequentially N-, C-, or both-terminal-residue-deleted 4EBP2 mutants were investigated by surface plasmon resonance (SPR) analysis. It was shown that the C-terminal His74-Glu89 sequence has an auxiliary, but indispensable, function in stabilizing the binding to eIF4E.

Binding preference of eIF4E for 4E-binding protein isoform and function of eIF4E N-terminal flexible region for interaction, studied by SPR analysis.

To investigate the binding preference of eIF4E for the three eIF4E-binding isoforms (4E-BP1-3) and the function of N-terminal flexible region of eIF4E for their interactions, the binding parameters of recombinant full-length and N-terminal residues-deleted eIF4Es with 4E-BP1-3 were investigated by the surface plasmon resonance (SPR) analysis. Consequently, it was clarified that 4E-BP2 exhibits the highest binding affinity for both m7GTP-bound and -unbound full-length eIF4Es when compared with 4E-BP1 and 4E-BP3. This is primarily due to the difference among their dissociation rates, because their association rates are almost the same.

Effect of N-terminal region of eIF4E and Ser65-phosphorylation of 4E-BP1 on interaction between eIF4E and 4E-BP1 fragment peptide.

To clarify the contribution of N-terminal region of eukaryotic initiation factor 4E (eIF4E) to the interaction with 4E-BP and to investigate the effect of 4E-BP phosphorylation on the interaction with eIF4E, the interaction profiles of the Ser65-unphosphorylated and phosphorylated peptides (Thr37-Thr70 fragment of 4E-BP1) with full-length and N-terminal 33 residues-deleted eIF4Es were investigated by fluorescence and SPR methods. The effect of N-terminal region of eIF4E on the interaction with 4E-BP1 peptides was shown to be dependent on the interaction state, that is, the steady-state fluorescence and kinetic-state SRP analyses showed the positive and negative contributions of the N-terminal region to the interaction with the peptide, respectively, despite its unphosphorylated or phosphorylated state. The comparison of the association constants of the peptide with those of full-length 4E-BP1 indicated the importance of N-terminal (1-36) and/or C-terminal (71-118) sequence of 4E-BP1 for the interaction, although the MD simulations suggested that the alpha-helical region (Arg56-Cys62) of 4E-BP1 peptide is sufficient for keeping the interaction.

Structural basis for mRNA Cap-Binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods.

Taking advantage of the Trp73 residue located close to the 4E-BP binding site of eIF4E, the interaction between the 4E-BP isoform and eIF4E was investigated by the Trp fluorescence titration method. Although no significant difference was observed among the association constants of three 4E-BP isoforms, the binding preference of 4E-BP2 over 4E-BP1 and -BP3 was shown, probably due to the effect of a 4E-BP2-specific LDRR (60-63) sequence for the binding with eIF4E. By contrast, surface plasmon resonance (SPR) analyses showed the binding preference of 4E-BP1, although the difference among the isoforms was also not significant.