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

Abstract

SUMMARY Binding of Ca2+-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 Ca2+ from the CaM C-lobe sites, even under high Ca2+ 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.

Signal Integration at Elongation Factor 2 Kinase

“…Apo-CaM and Ca 2ϩ /CaM Adopt a Common Mode of Binding at the C-lobe to Promote eEF-2K Activity toward eEF-2-We found that saturating apo-CaM induces a maximal eEF-2K activity against Pep-S to within 2-fold of Ca 2ϩ /CaM (Fig. 3F), suggesting that the final active conformations of auto-activated eEF-2K do not discriminate between these two structurally distinct forms of CaM when phosphorylating Pep-S. Our recent structure of the CBD ⅐ Ca 2ϩ -CaM complex (Protein Data Bank code 5J8H) suggests this may be due to a common binding mode of the Ca 2ϩ -loaded and Ca 2ϩ -free forms of CaM, where the CBD engages CaM mainly through the C-lobe, with engagement of the N-lobe (35) only occurring in the presence of Ca 2ϩ , thereby increasing the overall affinity.…”

“…Interestingly, the S500D/ W85S and S500E/W85S mutants are 10-and 5-fold more active than the W85S single mutant, respectively. Although not definitive, this may reflect our in vitro experiments demonstrating that Ser-500 phosphorylation greatly enhances the ability of CaM to activate eEF-2K, enough so as to partially rescue the activity of eEF-2K W85S, which binds CaM very weakly (35).…”

“…Additionally, to test the importance of CaM binding for the activity of the S500D or S500E mutants in cells, double mutants, which included a Trp-85 to Ser mutation in the CaM-binding site, were assessed. The W85S mutation significantly compromises the ability of eEF-2K to bind CaM as well as the activation of eEF-2K by various stimuli in cells (35). The S500D/W85S and S500E/W85S double mutants show a significantly decreased ability to phosphorylate eEF-2 compared with the S500D or S500E single mutants.…”

“…The N terminus of eEF-2K contains a CaM binding domain (CBD, ϳ79 -96) and a catalytic kinase domain (KD, ϳ116 -326). We solved the structure of the CBD ⅐ Ca 2ϩ -CaM (Protein Data Bank code 5J8H), and we found that the CBD engages the CaM C-lobe in an anti-parallel 1-5-8 mode through hydrophobic interactions reinforced by a pair of electrostatic contacts (35). A striking feature of this complex is the absence and presence of Ca 2ϩ from the C-and N-lobe sites of CaM, respectively, even under high Ca 2ϩ conditions, where Ca 2ϩ appears to enhance the CaM/eEF-2K interaction by promoting weak CaM N-lobe-mediated interactions.…”

Effect of Ca2+on the promiscuous target-protein binding mechanism of calmodulin

“…Both CaM dynamics and motif-dependent target-protein binding have been extensively studied via both experiments [2,8,[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] and simulations. Indeed, all-atom molecular dynamics (MD) simulations allow for a detailed description with molecular insights.…”