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

• Published in: Journal of molecular biology 2019-07, Vol.431 (15), p.2700-2717
• Main Authors: Piserchio, Andrea, Will, Nathan, Giles, David H., Hajredini, Fatlum, Dalby, Kevin N., Ghose, Ranajeet
• Format: Article
• Language: English
• Subjects: chemical shift perturbations; NMR structure; translational elongation; α-kinase
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2019.05.019

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. [Display omitted] •eEF-2K regulates translational elongation by phosphorylating eEF-2 resulting in the latter's reduced affinity for the ribosome.•The C-terminal region of eEF-2K has been predicted to contain the binding site for eEF-2.•We have determined the structure of a C-terminal fragment (eEF-2K562–725) of eEF-2K that encodes its last two helical repeats.•Using biochemical and biophysical analysis, we demonstrate that eEF-2K562–725 contains the key elements necessary for the eEF-2K/eEF-2 interaction.