Structure of the C-Terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase

Abstract

Eukaryotic elongation factor 2 kinase (eEF-2K) phosphorylates its only known physiological substrate, elongation factor 2 (eEF-2), which reduces the affinity of eEF-2 for the ribosome and results in an overall reduction in protein translation rates. The C-terminal region of eEF-2K, that is predicted to contain several SEL-1 like helical repeats (SLRs), is required for the phosphorylation of eEF-2. Using solution NMR methodology, we have determined the structure of a 99-residue fragment from the extreme C-terminus of eEF-2K (eEF-2K627-725) that encompasses a region previously suggested to be essential for eEF-2 phosphorylation. eEF-2K627-725 contains four helices, of which the first (αI) is flexible, and does not pack stably against the ordered helical core formed by the last three helices (αII-αIV). The helical core shows significant structural similarity with members of the tetratricopeptide repeat (TPR) family that includes SLRs. The two penultimate helices, αII and αIII, comprise the TPR, and the final helix, αIV, appears to have a capping function. The eEF-2K627-725 structure illustrates that the C-terminal deletion that was shown to abolish eEF-2 phosphorylation does so by destabilizing αIV and therefore, the helical core. Indeed, mutation of two conserved C-terminal tyrosines (Y712A/Y713A) in eEF-2K previously shown to abolish eEF-2 phosphorylation, leads to the unfolding of eEF-2K627-725. Preliminary functional analyses indicate that neither a peptide encoding a region deemed crucial for eEF-2 binding, nor isolated eEF-2K627-725 inhibit eEF-2 phosphorylation by full-length eEF-2K. Taken together, our data suggest that the extreme C-terminal region of eEF-2K, in isolation, does not provide a primary docking site for eEF-2.

Eukaryotic Elongation Factor 2 Kinase (eEF2K) in Cancer

“…The C-terminal part of eEF2K contains four predicted SEL1-like α-helical motifs; such motifs are often involved in protein–protein interactions [ 14 ] ( Figure 1 ). Although this C-terminal region and the extreme C-terminal end of eEF2K are required for it to phosphorylate eEF2, structural studies suggest that at least the last 99 amino acids do not provide a primary binding site for eEF2 [ 15 ]. At the extreme C-terminus is a short, highly conserved sequence that is critical for the ability of eEF2k to phosphorylate eEF2 [ 16 ].…”

Solution Structure of the Carboxy-Terminal Tandem Repeat Domain of Eukaryotic Elongation Factor 2 Kinase and Its Role in Substrate Recognition

“…The C-terminal region of eEF-2K has been predicted to contain the binding site for the substrate eEF-2 27 . In our earlier studies we had found that eEF-2K 627-725 was unable to inhibit the phosphorylation of eEF-2 by eEF-2K under our experimental conditions, leading us to conclude that the binding site of eEF-2 was not wholly contained within this region of eEF-2K 34 . We tested if the longer eEF-2K 562-725 construct was able to bind eEF-2 using a combination of NMR methods.…”

“…While S689 also deviates from Ala expected at this position, these two residue types share comparable steric profiles and helical propensities. The divergence of position 671 from the consensus (charged vs hydrophobic), was also noted in the structure of eEF-2K 627-725 , and it was found to not adversely impact the packing of helix B −1 with the A −1 and Cp helices 34 . However, due to the disorder in the nascent B −2 helix in the structure of eEF-2K 627-725 , this residue was found to be mostly solvent exposed.…”

“…We had previously solved the structure of the final C-terminal helical repeat of eEF-2K in the context of a shorter eEF-2K 627-725 fragment 34 . The overall architecture of the eEF-2K 562-725 fragment is quite similar to that of eEF-2K 627-725 when comparing the A −1 , B −1 and Cp helices only (backbone RMSD = 0.85 Å, Figure S6).…”

“…To this end, we previously determined the solution structure of the eEF-2K CBD in complex with Ca 2+ -calmodulin to define their primary interaction mode in atomic detail 33 . We had also solved the structure of the last C-terminal helical repeat that comprises the CTD 34 . In these continuing efforts, we present here the high-resolution structure of a larger C-terminal fragment encompassing residues G562-E725 (eEF-2K 562-725 ).…”