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Structural Features of Human Initiation Factor 4E, Studied by X-ray Crystal Analyses and Molecular Dynamics Simulations

2003; Elsevier BV; Volume: 328; Issue: 2 Linguagem: Inglês

10.1016/s0022-2836(03)00314-0

1089-8638

Koji Tomoo, Xu Shen, Koumei Okabe, Yoshiaki Nozoe, Shoichi Fukuhara, Shigenobu Morino, Masahiro Sasaki, Taizo Taniguchi, H. Miyagawa, Kunihiro Kitamura, Kin‐ichiro Miura, Toshimasa Ishida,

Carbohydrate Chemistry and Synthesis

The structural features of human eIF4E were investigated by X-ray crystal analyses of its cap analog (m7GTP and m7GpppA) 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 Å resolution revealed that the molecule forms a temple-bell-shaped surface of eight antiparallel β-structures, three α-helices and ten loop structures, where the N-terminal region corresponds to the handle of the bell. This concave backbone provides a scaffold for the mRNA cap-recognition pocket consisting of three receiving parts for the 5′-terminal m7G base, the triphosphate, and the second nucleotide. The m7G base is sandwiched between the two aromatic side-chains of Trp102 and Trp56. The two (m7G)NH–O (Glu103 carboxy group) hydrogen bonds stabilize the stacking interaction. The basic residues of Arg157 and Lys162 and water molecules construct a binding pocket for the triphosphate moiety, where a universal hydrogen-bonding network is formed. The flexible C-terminal loop region unobserved in the m7GTP complex was clearly observed in the m7GpppA complex, as a result of the fixation of this loop by the interaction with the adenosine moiety, indicating the function of this loop as a receiving pocket for the second nucleotide. On the other hand, MD simulation in an aqueous solution system revealed that the cap-binding pocket, especially its C-terminal loop structure, is flexible in the cap-free eIF4E, and the entrance of the cap-binding pocket becomes narrow, although the depth is relatively unchanged. SDS-PAGE analyses showed that this structural instability is highly related to the fast degradation of cap-free eIF4E, compared with cap-bound or 4E-BP/cap-bound eIF4E, indicating the conferment of structural stability of eIF4E by the binary or ternary complex formation. MD simulation of m7GpppA-bound Ser209-phosphorylated eIF4E showed that the size of the cap-binding entrance is dependent on the ionization state in the Ser209 phosphorylation, which is associated with the regulatory function through the switching on/off of eIF4E phosphorylation.