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.

Crystal structure and molecular dynamics simulation of ubiquitin-like domain of murine parkin.

Parkin is the gene product identified as the major cause of autosomal recessive juvenile Parkinsonism (AR-JP). Parkin, a ubiquitin ligase E3, contains a unique ubiquitin-like domain in its N-terminus designated Uld which is assumed to be a interaction domain with the Rpn 10 subunit of 26S proteasome. To elucidate the structural and functional role of Uld in parkin at the atomic level, the X-ray crystal structure of murine Uld was determined and a molecular dynamics simulation of wild Uld and its five mutants (K27N, R33Q, R42P, K48A and V56E) identified from AR-JP patients was performed.

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.

Structural features of human initiation factor 4E, studied by X-ray crystal analyses and molecular dynamics simulations.

The structural features of human eIF4E were investigated by X-ray crystal analyses of its cap analog (m(7)GTP and m(7)GpppA) complexes and molecular dynamics (MD) simulations of cap-free and cap-bound eIF4Es, as well as the cap-bound Ser209-phosphorylated eIF4E. Crystal structure analyses at 2.0 A resolution revealed that the molecule forms a temple-bell-shaped surface of eight antiparallel beta-structures, three alpha-helices and ten loop structures, where the N-terminal region corresponds to the handle of the bell.