Structural dynamics of the complex of calmodulin with a minimal functional construct of eukaryotic elongation factor 2 kinase and the role of Thr348 autophosphorylation

The calmodulin (CaM) activated alpha-kinase, eukaryotic elongation factor 2 kinase (eEF-2K), plays a central role in regulating translational elongation by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its ability to associate with the ribosome and suppressing global protein synthesis. Using TR (for truncated), a minimal functional construct of eEF-2K, and utilizing hydrogen/deuterium exchange mass spectrometry (HXMS), solution-state nuclear magnetic resonance (NMR) and biochemical approaches, we investigate the conformational changes accompanying complex formation between Ca2+-CaM and TR and the effects of autophosphorylation of the latter at Thr348, its primary regulatory site. Our results suggest that a CaM C-lobe surface, complementary to the one involved in recognizing the calmodulin-binding domain (CBD) of TR, provides a secondary TR-interaction platform. CaM helix F, which is part of this secondary surface, responds to both Thr348 phosphorylation and pH changes, indicating its integration into an allosteric network that encompasses both components of the Ca2+-CaM center dot TR complex. Solution NMR data suggest that CaMH107K, which carries a helix F mutation, is compromised in its ability to drive the conformational changes in TR necessary to enable efficient Thr348 phosphorylation. Biochemical studies confirm the diminished capacity of CaMH107K to induce TR autophosphorylation compared to wild-type CaM.

Automated summary

In this study, the conformational changes during complex formation between CaM-activated eEF-2K and TR, as well as the effects of TR autophosphorylation at Thr348, were investigated using a combination of HXMS, solution-state NMR, and biochemical approaches. The results suggest that the C-lobe surface of CaM interacts with TR and the helix F of CaM responds to Thr348 phosphorylation and pH changes, playing a crucial role in inducing conformational changes in TR for efficient phosphorylation.