Influence of Electric Charge Variation at Residues 209 and 159 on the Interaction of eIF4E with the mRNA 5‘ Terminus

Abstract

Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient protein synthesis in cap-dependent translation. The protein specifically binds the cap structure at the mRNA 5' terminus and facilitates the assembly of the mRNA with other initiation factors and the 40S ribosomal subunit. Phosphorylation of eIF4E is implicated in the regulation of the initiation step of translation. However, the molecular mechanism of this regulation still remains unclear. To address this problem, we have determined the binding affinities of eIF4E specifically mutated at position 209 or 159 for a series of novel mono- and dinucleotide cap analogues by a fluorometric time-synchronized titration method. A 1.5-3-fold reduction in the affinity of cap for the S209E mutant and a 1-2-fold increase in the affinity of cap for the S209K mutant, depending on the negative charge of phosphate chains, indicate that phosphorylation at Ser209 creates electrostatic repulsion between the protein and the negatively charged cap structure. The inhibition of the ability to bind cap analogues by the K159A mutant and its phosphorylated counterpart shows significant participation of Lys159 in the binding of the capped mRNA. Both structural modifications, phosphorylation and the replacement of lysine with alanine, result in an increase in the negative Gibbs free energy of association that is proportional to the length of the cap phosphate chain and additive, i.e., equal to the sum of the individual destabilizing changes of DeltaG degrees. The possible implication of these results for the mechanism of control of eIF4E by phosphorylation, especially for the "clamping model", is discussed.

The Source of the PB1 Gene in Influenza Vaccine Reassortants Selectively Alters the Hemagglutinin Content of the Resulting Seed Virus

“…In the absence of any difference in cellular vRNA and mRNA, the mechanism for altered amounts of HA protein is likely to act posttranscriptionally, altering viral protein translation. Upon influenza virus infection, the cellular translation initiation factor eIF4E, which recognizes the cap of mRNA, may become dephosphorylated and so host-cellular cap-dependent translation is inhibited (33)(34)(35)(36). Nevertheless, translation of the capped viral mRNA still occurs independently of eIF4E (37), and recently, it has been shown that the viral polymerase complex can act as a substitute for eIF4E (25).…”

Poisson–Boltzmann model analysis of binding mRNA cap analogues to the translation initiation factor eIF4E

“…Cap analogues, mutants of eIF4E, fluorescence titration measurements and experimental data Synthesis of cap analogues, mutagenesis, expression and purification of eIF4Es, as well as fluorescence measurements and data analysis were described in detail elsewhere [24].…”

“…The standard Gibbs free energy changes for complex formation were calculated from the K ass values according to the standard equation ∆G o = −RTlnK ass . The presented equilibrium binding constants for complexes of wild type and mutated eIF4E protein with m 7 GTP ligand were determined for the purpose of the present work, the remaining equilibrium binding constants connected to m 7 Gp 4 ligand were reported previously [24].…”

“…These structures are shown in Fig Analysis of electrostatic contributions to protein-ligand association energy requires that protonation states of their ionizable groups for given solution conditions are determined. The protonation probabilities of titrat-6 able groups in the eIF4E protein and terminal phosphate groups of m 7 GTP, m 7 Gp 4 ligands, corresponding to experimental conditions of the fluorescence titration measurements [24], were calculated as described in detail elsewhere [30,31], using the University of Houston Brownian Dynamics program (UHBD) [32,33]. For the purpose of predicting titration equilibria, all hydrogens were added and optimised with respect to their positions using CHARMM.…”

“…Besides standard titratable groups in proteins, also the terminal phosphate group of the ligands and the phosphate group of the Ser 209 (for their secondary titration) were treated as ionizable. All predicted protonation states refer to pH of 7.2, the same as that used in the experiments [24].…”