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Protein Sci. 2019 Dec;28(12):2089-2098. doi: 10.1002/pro.3753.

The role of calcium in the interaction between calmodulin and a minimal functional construct of eukaryotic elongation factor 2 kinase.

Lee K1,2, Kumar EA3, Dalby KN3,4, Ghose R1,2,5,6.

Author information

1
Department of Chemistry and Biochemistry, The City College of New York, New York, New York.
2
Graduate Program in Biochemistry, The Graduate Center of CUNY, New York, New York.
3
Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, Texas.
4
Graduate Program in Cell and Molecular Biology, University of Texas, Austin, Texas.
5
Graduate Program in Chemistry, The Graduate Center of CUNY, New York, New York.
6
Graduate Program in Physics, The Graduate Center of CUNY, New York, New York.

Abstract

Eukaryotic elongation factor 2 kinase (eEF-2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF-2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM-regulated kinases. We had previously identified a minimal construct of eEF-2K (TR) that is activated similarly to the wild-type enzyme by CaM in vitro and retains its ability to phosphorylate eEF-2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1-methyls of TR and Ile δ1- and Met ε-methyls of CaM, as probes of their mutual interaction and the influence of Ca2+ thereon. We find that in the absence of Ca2+ , CaM exclusively utilizes its C-terminal lobe (CaMC ) to engage the N-terminal CaM-binding domain (CBD) of TR in a high-affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca2+ -loaded N-terminal lobe of CaM (CaMN ) at increased Ca2+ levels serves to enhance the affinity further. These latter interactions under Ca2+ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca2+ -free state. Our data are consistent with a scenario in which Ca2+ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM-bound species without significantly modifying the conformation of TR within the final, active complex.

KEYWORDS:

calmodulin-regulated kinase; eukaryotic elongation factor 2 kinase (eEF-2K); methyl NMR spectroscopy; phosphorylation; translational regulation

PMID:
31626716
PMCID:
PMC6863733
[Available on 2020-12-01]
DOI:
10.1002/pro.3753

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