Friedland D E, Wooten W N B, LaVoy J E, Hagedorn C H, Goss D J
Department of Chemistry, Hunter College, and Graduate Center of the City University of New York, New York, New York 10021, USA.
Biochemistry. 2005 Mar 22;44(11):4546-50. doi: 10.1021/bi047645m.
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.
真核生物多亚基起始因子eIF4F是翻译机制的重要组成部分。翻译起始需要eIF4E识别mRNA的帽结构m(7)GpppN（其中N为任意核苷酸）。在此，我们比较了野生型eIF4E和高亲和力突变体eIF4E(K119A)与帽类似物和eIF4G肽的平衡及热力学结合特性。与野生型eIF4E相比，eIF4E(K119A)突变体与帽类似物的温度依赖性解离常数（K(d)）值明显更低，表明结合更紧密。虽然发现与帽类似物的相互作用是由焓驱动的，但熵的贡献也很显著。此外，eIF4E(K119A)对eIF4G肽的结合亲和力比对eIF4E(wt)的高2至4倍。这些结果表明，远离任一结合位点的点突变可同时改变对mRNA帽和eIF4G肽的结合亲和力。对结合的熵贡献表明，突变蛋白中的疏水相互作用更大，这很可能是由于构象变化所致。
