The eukaryotic initiation factor 4E (eIF4E) serves as a master switch that controls mRNA translation through the promotive binding to eIF4G and the regulative binding with the endogenous inhibitor 4E-BP. Although the bindings of eIF4G and 4E-BP to eIF4E proceed through the common eIF4E recognition Y(X)(4)L phi motif (X: variable, phi: hydrophobic) (first binding site), the relationship between their eIF4E binding mode and the functional difference is hardly known. Recently, we have clarified the existence and function of the second eIF4E binding site in 4E-BP. Surface plasmon resonance (SPR) analysis based on the sequential comparison between 4E-BP and eIF4GI clarified that eIF4G has the second binding site at the periphery of the (597)SDWL(601) sequence and that it plays an auxiliary but indispensable function in stabilizing the binding of the first binding sequence (572)YDREFLL(578). The kinetic parameters of the interactions of the eIF4GI and 4E-BP2 fragment peptides with eIF4E showed that the association (ka) and dissociation (kd) rates of the former peptide are about three and two orders of magnitude lower than those of the latter peptide, respectively. This means that eIF4G has a potent resistive property for release from eIF4E, although its rate of binding to eIF4E is not as high as that of 4E-BP, that is, 4E-BP is apt to bind to and be released from eIF4E, as compared with eIF4G. Isothermal titration calorimetry (ITC) showed the opposite behavior between the second binding sites of eIF4GI and 4E-BP for the interaction with eIF4E. This clearly indicates the importance of the second binding region for the difference in function between eIF4G and 4E-BP for eIF4E translation. (C) 2011 Elsevier Inc. All rights reserved.