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J Mol Biol. 2019 Jul 12;431(15):2700-2717. doi: 10.1016/j.jmb.2019.05.019. Epub 2019 May 18.

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

Author information

1
Department of Chemistry and Biochemistry, The City College of New York, NewYork, NY 10031, USA.
2
Department of Chemistry and Biochemistry, The City College of New York, NewYork, NY 10031, USA; Graduate Program in Biochemistry, The Graduate Center of CUNY, New York, NY 10016, USA.
3
Graduate Program in Cell and Molecular Biology, University of Texas, Austin, TX 78712, USA.
4
Graduate Program in Cell and Molecular Biology, University of Texas, Austin, TX 78712, USA; Division of Chemical Biology and Medicinal Chemistry, University of Texas, Austin, TX 78712, USA.
5
Department of Chemistry and Biochemistry, The City College of New York, NewYork, NY 10031, USA; Graduate Program in Biochemistry, The Graduate Center of CUNY, New York, NY 10016, USA; Graduate Program in Chemistry, The Graduate Center of CUNY, New York, NY 10016, USA; Graduate Program in Physics, The Graduate Center of CUNY, New York, NY 10016, USA. Electronic address: rghose@ccny.cuny.edu.

Abstract

Eukaryotic elongation factor 2 kinase (eEF-2K), an atypical calmodulin-activated protein kinase, regulates translational elongation by phosphorylating its substrate, eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome. The activation and activity of eEF-2K are critical for survival under energy-deprived conditions and is implicated in a variety of essential physiological processes. Previous biochemical experiments have indicated that the binding site for the substrate eEF-2 is located in the C-terminal domain of eEF-2K, a region predicted to harbor several α-helical repeats. Here, using NMR methodology, we have determined the solution structure of a C-terminal fragment of eEF-2K, eEF-2K562-725 that encodes two α-helical repeats. The structure of eEF-2K562-725 shows signatures characteristic of TPR domains and of their SEL1-like sub-family. Furthermore, using the analyses of NMR spectral perturbations and ITC measurements, we have localized the eEF-2 binding site on eEF-2K562-725. We find that eEF-2K562-725 engages eEF-2 with an affinity comparable to that of the full-length enzyme. Furthermore, eEF-2K562-725 is able to inhibit the phosphorylation of eEF-2 by full-length eEF-2K in trans. Our present studies establish that eEF-2K562-725 encodes the major elements necessary to enable the eEF-2K/eEF-2 interactions.

KEYWORDS:

NMR structure; chemical shift perturbations; translational elongation; α-kinase

PMID:
31108082
PMCID:
PMC6599559
[Available on 2020-07-12]
DOI:
10.1016/j.jmb.2019.05.019

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