Abstract
mRNA 5′-cap recognition by the eukaryotic translation initiation factor eIF4E 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 eIF4E for 20 different cap analogues cover six orders of magnitude; with the highest affinity observed for m
7GTP (∼1.1×10
8
M
−1). The affinity of the cap analogues for eIF4E 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 α, β, γ 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
7G/Trp56 configuration is a precondition to form three hydrogen bonds with Glu103 and Trp102. Electrostaticly steered eIF4E-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
7GTP–eIF4E binding, which results from dominant charge-related interactions (Δ
H°=−17.8
kcal/mol, Δ
S°=−23.6
cal/mol
K). For recruitment of synthetic eIF4GI, eIF4GII, and 4E-BP1 peptides to eIF4E, all the association constants were ∼10
7
M
−1, in decreasing order: eIF4GI>4E-BP1>eIF4GII∼4E-BP1(P-Ser65) ∼4E-BP1(P-Ser65/Thr70). Phosphorylation of 4E-BP1 at Ser65 and Thr70 is insufficient to prevent binding to eIF4E. Enhancement of the eIF4E affinity for cap occurs after binding to eIF4G peptides.