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The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 A resolution (where 1 A=0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein-eIF4E phosphorylation.

The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5′-triphosphate (m7GTP) and P1-7-methylguanosine-P3-adenosine-5′,5′-triphosphate (m7GpppA)] were determined at 2.0Å resolution (where 1Å = 0.1nm). The flexibility of the C-terminal loop region of eIF4E complexed with m7GTP was significantly reduced when complexed with m7GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein—eIF4E phosphorylation.

Recombinant human eukaryotic initiation factor-4E (eIF-4E), purified by m7GTP-Sepharose 4B affinity chromatography, was used for crystallization. After concentration of the eIF-4E protein (7 mg/ml), the solution was subjected to crystallization by the hanging-drop method. Transparent needle crystals complexed with m7GTP were obtained from 50 mM 2-(N-morpholino)ethanesulfonic acid-KOH buffer (pH 6.5) containing 25% (w/v) polyethylene glycol 6000 and 0.2 M (NH4)2SO4. The crystals belong to tetragonal space group P4(1) or P4(3), of Z = 4, with unit-cell dimensions of a = 89.26, b = 89.26, and c = 38.51 angstrum, and diffract beyond 2.1 angstrum resolution. The Vm value was calculated to be 3.07 angstrum 3/Da, which indicates a solvent content of 59.9%.

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