ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin

Abstract

Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase eukaryotic elongation factor 2 (eEF-2) resulting from its specific phosphorylation by the calmodulin (CaM)-activated α-kinase eEF-2 kinase (eEF-2K). It has been suggested that the eEF-2K response to reduced cellular energy levels is indirect and mediated by the universal energy sensor AMP-activated protein kinase (AMPK) through direct stimulatory phosphorylation and/or downregulation of the eEF-2K-inhibitory nutrient-sensing mTOR pathway. Here, we provide structural, biochemical, and cell-biological evidence of a direct energy-sensing role of eEF-2K through its stimulation by ADP. A crystal structure of the nucleotide-bound complex between CaM and the functional core of eEF-2K phosphorylated at its primary stimulatory site (T348) reveals ADP bound at a unique pocket located on the face opposite that housing the kinase active site. Within this basic pocket (BP), created at the CaM/eEF-2K interface upon complex formation, ADP is stabilized through numerous interactions with both interacting partners. Biochemical analyses using wild-type eEF-2K and specific BP mutants indicate that ADP stabilizes CaM within the active complex, increasing the sensitivity of the kinase to CaM. Induction of energy stress through glycolysis inhibition results in significantly reduced enhancement of phosphorylated eEF-2 levels in cells expressing ADP-binding compromised BP mutants compared to cells expressing wild-type eEF-2K. These results suggest a direct energy-sensing role for eEF-2K through its cooperative interaction with CaM and ADP.

Keywords: alpha-kinase; calmodulin; protein translation; serine/threonine kinase.

Fig. 1.

Structure of the nucleotide bound (NB) CaM•peEF-2K_TR complex. (A) The NB structure is shown in ribbon representation; the CaM-targeting motif (CTM, cyan), the kinase domain (KD, lime), and the C-terminal domain (CTD, orange) are highlighted. Also shown is the C-terminal (CaM_C, dull yellow) lobe of CaM; the dynamic N-terminal lobe of CaM (CaM_N) is not resolved in the structure. The nucleotides, ATP bound at the kinase active site, and ADP bound at the basic pocket (BP) are depicted as spheres. (B) Key interactions of ATP bound at the peEF-2K_TR active site are illustrated with the peEF-2K_TR sidechains colored brown. The positions of selected sidechains, of the nucleotide-free (NF, in yellow) complex, whose conformations are altered (indicated by the cyan ovals and arrows) upon ATP binding, are shown together with the sidechains of key active site residues (labeled in the lighter font). (C) The P- and the β10-αD loops in the NB complex (brown) are slightly more closed (indicated by the red arrows) relative to the N/D-loop compared to that in the NF complex (yellow). The bound ATP is shown in stick representation.

Fig. 2.

Interactions that stabilize ADP at the BP in the NB complex. (A) Key interactions that stabilize ADP at the BP are indicated. The sidechains of peEF-2K_TR and CaM are colored brown and cyan, respectively, with the corresponding residues labeled using 1-letter and 3-letter codes. (B) Conformational differences between the NF (peEF-2K_TR in yellow, CaM in pink) and NB (peEF-2K_TR in brown and CaM in cyan) complexes at the BP and adjoining regions are shown. The key CTM anchor residue W85 is indicated for reference; ADP is shown in stick representation. The β6—β7 loop is also indicated with its incomplete chain trace in the NF structure denoted by the ‘*’. The right panel shows an expansion of the region indicated by the black rectangle on the left panel. Regions that show significant local differences between the NF and NB structures are indicated by the red arrows. The sidechains of F155 and Arg90 (both labeled in red), which are only partially resolved in the NF structure (suggesting disorder), become ordered in the NB complex. The ADP molecule is omitted for visual ease.

Fig. 3.

Influence of ADP on the CaM-mediated activity wild-type eEF-2K. (A) A representative thermogram for the interaction of ADP with the CaM•eEF-2K complex is shown. The active site was blocked using the ATP-competitive inhibitor A484954. The K_D represents the value obtained by a fit to a single site model (the stoichiometric constant was fixed to 1) for the specific thermogram shown. (B) Variation of the CaM sensitivity (K_CaM) of the activity of wild-type peEF-2K against the Soxtide substrate with ADP concentration (C_ADP) is shown. Circles and error bars indicate the average and SD values, respectively, obtained from fits of the observed phosphorylation rates (k_obs) with CaM concentration at a fixed C_ADP value (SI Appendix, Fig. S9A). The red lines indicate 95% confidence bounds of the fit. (C–F) Changes in k_obs with CaM concentration in the absence (filled circles) or in the presence of a saturating amount of ADP (900 μM; open circles). Data are shown for (C) full-length wild-type (WT) peEF-2K and the corresponding BP mutants: (D) R114A, (E) R116A, and (F) H230Q. The circles and error bars indicate mean values and corresponding SDs over two replicates for each data point.

Fig. 4.

eEF-2 phosphorylation in cells expressing wild-type eEF-2K or specific BP mutants. (A) eEF-2K^−/− MCF10A cells expressing wild-type eEF-2K (WT) or specific BP mutants (R114A or R116A) were assessed for eEF-2K activity through eEF-2 phosphorylation (on Thr-56) levels following no treatment (NT; control), starvation with DPBS (STRV), or incubation with the glycolysis inhibitor, 2-deoxyglucose (2-DG). A representative immunoblot is shown on the top panel. Pan-actin is used as loading control. (B) eEF-2 phosphorylation levels in cells expressing wild-type eEF-2K or the R114A or R116A mutants under specific conditions are indicated. Data are normalized to the untreated WT eEF-2K sample. Data represent three independent experiments (N = 3); all data points are shown; the corresponding mean and SDs are indicated by the horizontal and vertical lines, respectively. Statistical significance, obtained through a two-way ANOVA and Tukey post hoc analysis, for relevant pairs are indicated. P value markers: < 0.0001 (****), 0.0001 to 0.001 (***), 0.001 to 0.01 (**), 0.01 to 0.05 (*).