The critical role of the C-terminal lobe of calmodulin in activating eukaryotic elongation factor 2 kinase

Eukaryotic elongation factor 2 kinase (eEF-2K), a member of the α-kinase family, modulates translational rates by phosphorylating eEF-2, a GTPase that facilitates the translocation of the nascent chain on the ribosome during the elongation phase of protein synthesis. eEF-2K is regulated by diverse cellular cues, many of which sensitize it to the Ca²⁺-effector protein calmodulin (CaM). CaM, which binds and allosterically activates eEF-2K in the presence of Ca²⁺, contains two structural "lobes," each with a pair of Ca²⁺-binding EF hands. Using kinetic analysis, we demonstrate that the isolated C-terminal lobe of CaM (CaM_C) is sufficient to engage and fully activate eEF-2K in a Ca²⁺-dependent fashion. Genetically fusing CaM_C to the N terminus of eEF-2K, upstream of its critical CaM-targeting motif via a flexible 2-glycine linker, results in a chimeric species (CaM_C is linked to N-truncated eEF-2K [C-LiNK]) that is constitutively active independent of external CaM and Ca²⁺. A structure of the C-LiNK functional core reveals no substantial deviation in the overall conformations of the structural modules and orientations of key catalytic-site residues relative to the heterodimeric complex between full-length CaM and eEF-2K. These observations demonstrate that, in contrast to other CaM-regulated kinases, CaM_C alone is sufficient to activate eEF-2K fully. The proximity effect of CaM_C in the context of C-LiNK removes the requirement for external Ca²⁺, whose apparent role is to enhance the CaM affinity of eEF-2K and drive kinase activation. Further, the responsiveness of eEF-2K to regulatory stimuli in cells appears to be lost in C-LiNK, presumably due to its permanently "on" state.