Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the α-kinase family of atypical serine/threonine kinases, phosphorylates eEF-2 to slow ribosomal translocation and modulate translational elongation. eEF-2K activation requires Ca²⁺/calmodulin (CaM) and integrates upstream signals through specific sites within an intrinsically disordered regulatory loop (R-loop; ∼321-520) that links the α-kinase core to a C-terminal domain. Unlike canonical CaM-dependent kinases that are activated by displacement of an autoinhibitory segment that occludes the active site, eEF-2K is activated by CaM-driven stabilization of an active state; Ca²⁺/CaM engagement triggers rapid autophosphorylation at T348, which is essential for full activity. Phosphorylation on S500, by eEF-2K or PKA, lowers the CaM requirement (∼20-fold) without increasing maximal catalytic turnover. Here we show that the phosphomimetic S500D markedly enhances CaM binding in the T348-phosphorylated enzyme. S500D also elevates CaM-independent ("intrinsic") activity even in the absence of phosphorylation at T348, although maximal activity requires modification at both sites. Hydrogen-deuterium exchange mass spectrometry reveals CaM-dependent conformational changes near S500, consistent with relief of inhibitory constraints. Deletion of residues near S500 mimics S500D, increasing intrinsic activity and CaM binding in vitro and enhancing eEF-2 phosphorylation in cells, supporting an inhibitory role for this segment. Prior studies have linked S500 phosphorylation to eEF-2K degradation, suggesting a dual regulatory role. We demonstrate that phosphorylation at T348 and S500 synergize to stabilize an active-like conformation and increase CaM responsiveness, effectively lowering the Ca²⁺/CaM threshold for eEF-2K activation and enabling the integration of Ca²⁺, cAMP/PKA, and metabolic cues.