AbstractUniversity of ManchesterShirley TaitMaster of PhilosophyInteractions on the Dorsal Surface of eIF4E2010Eukaryotic initiation factor 4E (eIF4E) is the messenger RNA cap binding protein, which recruits eIF4G and eIF4A allowing translation initiation to proceed. The eIF4E binding proteins (4E-BPs) are small, heat-stable proteins (~12KDa) that act as repressors of translation. They function by binding the same site as eIF4G on the dorsal region of eIF4E, opposite the cap binding site, thereby competing for the site with eIF4G and inhibiting translation. Phosphorylation of the 4E-BPs in response to growth factors, hormones, mitogens and cytokines causes a reduction in binding to eIF4E, allowing eIF4G to bind and translation initiation to proceed. eIF4E binding protein 1 (4E-BP1) has been shown to be unstructured in solution and folds into a helical conformation on binding eIF4E, but what predisposes the 4E-BP1 to fold in the correct conformation on the dorsal surface of eIF4E?A number of residues of both eIF4E and 4E-BP1 have been identified which are thought to be important in the interaction between the proteins. However the role of solvent facing residues aspartic acid 55, lysine 57, glutamic acid 61 and arginine 63 in the -helical stretch of the eIF4E binding motif of 4E-BP1 that are not directly involved in binding eIF4E was still unexplained.In this study a number of techniques have been employed to try and further our understanding of the role of folding in determining the affinity between 4E-BP1 and the dorsal face of eIF4E and thus the ability of 4E-BP1 to act as an inhibitor of translation. Isothermal titration calorimetry was used to determine binding affinities between peptides of 4E-BP151-67 and recombinant eIF4E along with circular dichroism and NMR studies on the peptides in solution and titrated with recombinant eIF4E. These residues were identified as contributing to the propensity of the 4E-BP1 to fold into the correct conformation for binding to eIF4E.So how does phosphorylation of 4E-BP1 cause a reduction in binding to eIF4E? A number of possibilities have been put forward. One explanation is that phosphorylation of 4E-BP1 could lead to electrostatic repulsion against a glutamic acid residue on eIF4E. Another possibility is that the close proximity of the phosphorylated serine on 4E-BP1 to this glutamic acid on eIF4E and glutamic acid 61 of the 4E-BP1 itself could lead to destabilisation of the -helix within the binding motif. However the role of phosphorylation of the 4E-BPs in terms of structural effects on the region of their eIF4E binding motif has remained uncharacterised.Isothermal titration calorimetry and NMR experiments in this study indicate that phosphorylation of serine 65 at the C-terminal end of the helical region of 4E-BP1 can affect the disorder-order transition to favour the unfolded form that cannot bind eIF4E. This adds to the growing data on unstructured proteins and can give insight into the mechanisms employed by this group of proteins.
|Date of Award||31 Dec 2010|
- The University of Manchester
|Supervisor||John Mccarthy (Supervisor)|