7-methylguanosine-5--diphosphate and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

The mRNA cap-binding protein eIF4E is the limiting factor in the eIF4F translation initiation complex, which mediates the binding of the 40S ribosome to the mRNA. 15N relaxation studies have been used to characterize the backbone dynamics of deuterated eIF4E in a CHAPS micelle for the apoprotein, the m7GDP-bound form, and the dinucleotide (m7GpppA)-bound form, as well as for CHAPS-free eIF4E. Large differences in overall correlation time between the CHAPS-free form (11.8 ns) and samples containing different concentrations of CHAPS (15.9-19.4 ns) indicate that eIF4E is embedded in a large micelle in the presence of CHAPS, with a total molecular weight in the range of 40-60 kDa. CHAPS seems to restrict the mobility of the a2-b3 and a4-b5 loops which are thought to be embedded in the micelle. No significant changes in overall mobility were seen between the m7 GDP-bound form, the m7GpppA-bound form, and the apoprotein. Amide hydrogen exchange data indicate the presence of slowly exchanging amides in two surface-exposed helices (a2 and a4), as well as the a4-b5 loop, indicating protection by the CHAPS micelle. The micelle covers the convex side of the protein away from the cap-binding site.

Topics: Cholic Acids; Cloning, Molecular; Detergents; Eukaryotic Initiation Factor-4E; Guanosine Diphosphate; Hydrogen Bonding; Kinetics; Micelles; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; Peptide Initiation Factors; Protein Structure, Secondary; Recombinant Proteins; RNA Cap Analogs; RNA Caps; Saccharomyces cerevisiae

eIF4E, the mRNA cap binding protein, is a master switch that controls eukaryotic translation. To be active, it must bind eIF4G and form the eIF4F complex, which also contains eIF4A. Translation is downregulated by association of eIF4E with 4E-BP, which occupies the eIF4G binding site. Signalling events acting on 4E-BP cause it to dissociate from eIF4E, and eIF4E is then free to bind eIF4G to form the active eIF4F complex. We have solved the structure of the yeast eIF4E/m7Gpp complex in a CHAPS micelle. We determined the position of the second nucleotide in a complex with m7GpppA, and identified the 4E-BP binding site. eIF4E has a curved eight-stranded antiparallel beta-sheet, decorated with three helices on the convex face and three smaller helices inserted in connecting loops. The m7G of the cap is intercalated into a stack of tryptophans in the concave face. The 4E-BP binding site is located in a region encompassing one edge of the beta-sheet, the adjacent helix a2 and several regions of non-regular secondary structure. It is adjacent to, but does not overlap the cap-binding site.

Topics: Amino Acid Sequence; Binding Sites; Carrier Proteins; Cholic Acids; Detergents; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factors; Guanosine Diphosphate; Magnetic Resonance Spectroscopy; Micelles; Molecular Sequence Data; Peptide Initiation Factors; Protein Conformation; RNA Cap Analogs; Tryptophan; Yeasts