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. This concave backbone provides a scaffold for the mRNA cap-recognition pocket consisting of three receiving parts for the 5'-terminal m(7)G base, the triphosphate, and the second nucleotide. The m(7)G base is sandwiched between the two aromatic side-chains of Trp102 and Trp56. The two (m(7)G)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 m(7)GTP complex was clearly observed in the m(7)GpppA 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 m(7)GpppA-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.
Tomoo
Koji
K
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. tomoo@gly.oups.ac.jp
Shen
Xu
X
Okabe
Koumei
K
Nozoe
Yoshiaki
Y
Fukuhara
Shoichi
S
Morino
Shigenobu
S
Sasaki
Masahiro
M
Taniguchi
Taizo
T
Miyagawa
Hiroo
H
Kitamura
Kunihiro
K
Miura
Kin-ichiro
K
Ishida
Toshimasa
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D039561:Eukaryotic Initiation Factor-4E / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D012314:RNA Cap Analogs / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012694:Serine / Q000737:chemistry; D055672:Static Electricity; D013816:Thermodynamics
D039561:Eukaryotic Initiation Factor-4E; D046911:Macromolecular Substances; D012314:RNA Cap Analogs; D011994:Recombinant Proteins; D012694:Serine