Positive Heat Capacity Change upon Specific Binding of Translation Initiation Factor eIF4E to mRNA 5‘ Cap

Abstract

Specific recognition of the mRNA 5‘ cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry were used to examine the thermodynamics of eIF4E binding to a cap-analogue, 7-methylGpppG. A van't Hoff plot revealed nonlinearity characterized by an unexpected, large positive molar heat capacity change ( = +1.92 ± 0.93 kJ·mol^-1·K^-1), which was confirmed by direct ITC measurements ( = +1.941 ± 0.059 kJ·mol^-1·K^-1). This unique result appears to come from an extensive additional hydration upon binding and charge-related interactions within the binding site. As a consequence of the positive , the nature of the thermodynamic driving force changes with increasing temperature, from enthalpy-driven and entropy-opposed, through enthalpy- and entropy-driven in the range of biological temperatures, into entropy-driven and enthalpy-opposed. Comparison of the van't Hoff and calorimetric enthalpy values provided proof for the ligand protonation at N(1) upon binding, which is required for tight stabilization of the cap−eIF4E complex. Intramolecular self-stacking of the dinucleotide cap-analogue was analyzed to reveal the influence of this coupled process on the thermodynamic parameters of the eIF4E−mRNA 5‘ cap interaction. The temperature-dependent change in the conformation of 7-methylGpppG shifts significantly the intrinsic = −72.9 ± 4.2 kJ·mol^-1 and = −116 ± 58 J·mol^-1·K^-1 of binding to the less negative resultant values, by = +9.76 ± 1.15 kJ·mol^-1 and = +24.8 ± 2.1 J·mol^-1·K^-1 (at 293 K), while the corresponding = −0.0743 ± 0.0083 kJ·mol^-1·K^-1 is negligible in comparison with the total .