title: A mutant of eukaryotic protein synthesis initiation factor eIF4E(K119A) has an increased binding affinity for both m7G cap analogues and eIF4G peptides.
abstract: The eukaryotic multisubunit initiation factor eIF4F is an essential component of the translational machinery. Recognition of the cap structure of mRNA, m(7)GpppN, where N is any nucleotide, by eIF4E is required for initiation of translation. Here we compare the equilibrium and thermodynamic binding characteristics of wild-type eIF4E and a high-affinity mutant, eIF4E(K119A), with those of cap analogues and eIF4G peptides. The temperature-dependent K(d) values for cap analogues were markedly lower, indicating tighter binding, with the eIF4E(K119A) mutant compared with wild-type eIF4E. Although interactions with cap analogues were found to be enthalpically driven, entropic contributions were also significant. Moreover, the binding affinities of eIF4G peptides were 2-4-fold tighter for eIF4E(K119A) than for eIF4E(wt). These results demonstrate that the binding affinity for both the mRNA cap and eIF4G peptides can be simultaneously altered by point mutations distant from either binding site. Entropic contributions to binding suggesting hydrophobic interactions are larger in the mutant protein and are most likely due to a conformational change.
authors.affiliation: Department of Chemistry, Hunter College, and Graduate Center of the City University of New York, New York, New York 10021, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Friedland
authors.forename: W N B
authors.initials: WN
authors.lastname: Wooten
authors.forename: J E
authors.initials: JE
authors.lastname: LaVoy
authors.forename: C H
authors.initials: CH
authors.lastname: Hagedorn
authors.forename: D J
authors.initials: DJ
authors.lastname: Goss
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics* / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism*; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012314:RNA Cap Analogs / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
chemical_list: C113920:EIF4G1 protein, human; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D020033:Protein Isoforms; D012314:RNA Cap Analogs; D006151:Guanosine; C016578:7-methylguanosine; D008239:Lysine; D000409:Alanine