Biophysical studies of eIF4E cap-binding protein:: Recognition of mRNA 5′ cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins

mRNA 5'-cap recognition by the eukaryotic translation initiation factor elF4E has been exhaustively characterized with the aid of a novel fluorometric, time-synchronized titration method, and X-ray crystallography. The association constant values of recombinant elF4E for 20 different cap analogues cover six orders of magnitude; with the highest affinity observed for m(7)GTP (similar to 1.1 x 10(8) M-1). The affinity of the cap analogues for elF4E correlates with their ability to inhibit in vitro translation. The association constants yield contributions of non-covalent interactions involving single structural elements of the cap to the free energy of binding, giving a reliable starting point to rational drug design. The free energy of 7-methylguanine stacking and hydrogen bonding (- 4.9 kcal / mol) is separate from the energies of phosphate chain interactions (- 3.0, - 1.9, - 0.9 kcal/mol for alpha, beta, gamma phosphates, respectively), supporting two-step mechanism of the binding. The negatively charged phosphate groups of the cap act as a molecular anchor, enabling further formation of the intermolecular contacts within the cap-binding slot. Stabilization of the stacked Trp102/m(7)G/Trp56 configuration is a precondition to form three hydrogen bonds with Glu103 and Trp102. Electrostaticly steered elF4E-cap association is accompanied by additional hydration of the complex by approximately 65 water molecules, and by ionic equilibria shift. Temperature dependence reveals the enthalpy-driven and entropy-opposed character of the m(7)GTP-eIF4E binding, which results from dominant charge-related interactions (DeltaH(o) = - 17.8 kcal/mol, DeltaS(o) = - 23.6 cal/ mol K). For recruitment of synthetic eIF4GI, eIF4GII, and 4E-BP1 peptides to elF4E, all the association constants were - 10(7) M-1, in decreasing order: eIF4GI > 4E-BP1 > elF4GII similar to 4E-BP1 (P-Ser65) similar to 4E-BP1 (P-Ser65/Thr70). Phosphorylation of 4E-BP1 at Ser65 and Thr70 is insufficient to prevent binding to elF4E. Enhancement of the elF4E affinity for cap occurs after binding to eIF4G peptides. (C) 2002 Elsevier Science Ltd. All rights reserved.