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. 2016 Sep 6;24(9):1441-51.
doi: 10.1016/j.str.2016.06.015. Epub 2016 Aug 4.

Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin

Free PMC article

Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin

Kwangwoon Lee et al. Structure. .
Free PMC article


Binding of Ca(2+)-loaded calmodulin (CaM) activates eukaryotic elongation factor 2 kinase (eEF-2K) that phosphorylates eEF-2, its only known cellular target, leading to a decrease in global protein synthesis. Here, using an eEF-2K-derived peptide (eEF-2KCBD) that encodes the region necessary for its CaM-mediated activation, we provide a structural basis for their interaction. The striking feature of this association is the absence of Ca(2+) from the CaM C-lobe sites, even under high Ca(2+) conditions. eEF-2KCBD engages CaM largely through the C lobe of the latter in an anti-parallel 1-5-8 hydrophobic mode reinforced by a pair of unique electrostatic contacts. Sparse interactions of eEF-2KCBD with the CaM N lobe results in persisting inter-lobe mobility. A conserved eEF-2K residue (W85) anchors it to CaM by inserting into a deep hydrophobic cavity within the CaM C lobe. Mutation of this residue (W85S) substantially weakens interactions between full-length eEF-2K and CaM in vitro and reduces eEF-2 phosphorylation in cells.


Figure 1
Figure 1. Schematic representation of the domain organization of eEF-2K
The catalytic domain (CD, cyan), the basic allosteric pocket (yellow) and the critical auto-phosphorylation site T348 (green) on the regulatory loop (R-loop) are shown; the C-terminal domain (CTD, red) contains multiple Sel1 like repeats (Mittl and Schneider-Brachert, 2007). The N-terminal CaM-binding domain (CBD, 74-100, blue) is encoded by the eEF-2KCBD peptide used here.
Figure 2
Figure 2. Interaction between eEF-2KCBD with CaM in the presence of Ca2+
(A) ITC trace representative of three repeats is shown. For the trace shown, KD=137.2±18.8 nM, ΔH=−16.6 KCal•mol−1, ΔS=−22.5 Cal•mol−1•K−1. (B) Backbone amide chemical shift perturbations induced on Ca2+-CaM upon formation of the 1:1 Ca2+-CaM•eEF-2KCBD complex. The locations of the eight helices (A, B, C, D on the N-lobe and E, F, G, H on the C-lobe) are shown. The blue bar represents residues that are unassigned in the Ca2+-CaM•eEF-2KCBD complex.
Figure 3
Figure 3. Steady-state {1H}-15N NOE for eEF-2KCBD in the Ca2+-CaM•eEF-2KCBD complex
The NOEs for the sidechain indole NH positions for W85 and W99 are shown in red. The backbone amide resonance of H80 was not detected in the spectra and is depicted by the green ‘*’. The locations of the two prolines are shown in blue.
Figure 4
Figure 4. Structural differences in CaM in the presence of eEF-2KCBD under low and high Ca2+ conditions
(A) Chemical shift changes induced on apo-CaM in the presence of a 3-fold molar ratio of eEF-2KCBD. The black and green bars represent residues that are unassigned in the apo-CaM•eEF-2KCBD complex and apo-CaM alone, respectively. (B) Differences in chemical shifts between the apo-CaM•eEF-2KCBD and Ca2+-CaM•eEF-2KCBD complexes. The blue and green bars represent residues that are unassigned in the Ca2+-CaM•eEF-2KCBD complex and the apo-CaM•eEF-2KCBD complex, respectively.
Figure 5
Figure 5. Solution structure of the Ca2+-CaM•eEF-2KCBD complex
20 structures representing the final NMR ensemble have been overlaid using the C-lobe and eEF-2KCBD (see Table 1). The N-lobe (1–77) and C-lobe of CaM (82–148) have been colored green and blue respectively and the linker (78–81) is colored grey. eEF-2KCBD is colored dark orange. The N-lobe of CaM, the linker, 6 residues on the N-terminus and 7 residues on the C-terminus of eEF-2KCBD are hidden to allow better visualization of the C-lobe eEF-2KCBD interactions in the lower panel. The sidechains of the critical W85 are shown and colored light orange. The eEF-2KCBD residues are in italics and shown in orange. The Ca2+ ions occupying the N-lobe sites are not shown.
Figure 6
Figure 6. Contacts between CaM and eEF-2KCBD
(A) The CaM/eEF-2KCBD interface is stabilized by three sets of interactions: 1-5-8 hydrophobic docking (residues labeled and outlined using blue rectangles) of eEF-2KCBD onto the the CaM C-lobe; interactions between P98 (outlined using a green rectangle) on eEF-2KCBD and the CaM N-lobe; two sets of electrostatic interactions (outlined by red circles) involving basic residues on eEF-2KCBD and acidic residues on CaM. Detailed views of the 1-5-8 binding (B) and the interaction of P98 (C) with the C-lobe and the N-lobe of CaM, respectively, are shown. Locations of key residues on the CaM surface are colored (yellow for the C-lobe and magenta for the N-lobe) and labeled (in black). eEF-2KCBD residues are labeled in orange. The dashed lines indicate residues that point out the plane of the paper.
Figure 7
Figure 7. Influence of the W85S mutation on full-length eEF-2K
(A) Fluorescence binding assays measuring interaction between wild-type full-length eEF-2K (blue), eEF-2KCBD (black) or the W85S mutant (red) and dansyl-CaM in the presence of Ca2+. The points (errors from duplicate measurements) represent the fraction of CaM in the bound state (calculated using Equation 5) at various concentrations of titrant and the solid curves represent the fits to Equation 6. (B) Results of the Western blot analysis for eEF-2 phosphorylated on T56 after transfection of knockout MCF-10A (eEF-2K−/−) cells with vectors encoding wild-type eEF-2K or the W85S mutant along with appropriate controls. Also shown is the level of eEF-2 phosphorylated on T56 upon stimulation with 25 mM 2-DOG (30 min) (C), 400 μM H2O2 (1 h) (D), 5 μM ionomycin (5 min) or starvation with DPBS (6 h) (E), along with the appropriate controls. Note that the blots shown in (B) have been cropped from those shown in (D) for untreated samples.

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