Crystallographic and Mass Spectrometric Characterisation of eIF4E with N7-alkylated Cap Derivatives

Structural complexes of the eukaryotic translation initiation factor 4E (eIF4E) with a series of N7-alkylated guanosine derivative mRNA cap analogue structures have been characterised. Mass spectrometry was used to determine apparent gas-phase equilibrium dissociation constants (Kd) values of 0.15 μM, 13.6 μM, and 55.7 μM for eIF4E with 7-methyl-GTP (m7GTP), GTP, and GMP, respectively. For tight and specific binding to the eIF4E mononucleotide binding site, there seems to be a clear requirement for guanosine derivatives to possess both the delocalised positive charge of the N7-methylated guanine system and at least one phosphate group. We show that the N7-benzylated monophosphates 7-benzyl-GMP (Bn7GMP) and 7-(p-fluorobenzyl)-GMP (FBn7GMP) bind eIF4E substantially more tightly than non-N7-alkylated guanosine derivatives (Kd values of 7.0 μM and 2.0 μM, respectively). The eIF4E complex crystal structures with Bn7GMP and FBn7GMP show that additional favourable contacts of the benzyl groups with eIF4E contribute binding energy that compensates for loss of the β and γ-phosphates. The N7-benzyl groups pack into a hydrophobic pocket behind the two tryptophan side-chains that are involved in the cation–π stacking interaction between the cap and the eIF4E mononucleotide binding site. This pocket is formed by an induced fit in which one of the tryptophan residues involved in cap binding flips through 180° relative to structures with N7-methylated cap derivatives. This and other observations made here will be useful in the design of new families of eIF4E inhibitors, which may have potential therapeutic applications in cancer.