The Molecular Mechanism of Eukaryotic Elongation Factor 2 Kinase Activation [Signal Transduction]

July 10th, 2014 by Tavares, C. D. J., Ferguson, S. B., Giles, D. H., Wang, Q., Wellmann, R. M., O'Brien, J. P., Warthaka, M., Brodbelt, J. S., Ren, P., Dalby, K. N.

Calmodulin (CaM)-dependent elongation factor 2 kinase (eEF-2K) impedes protein synthesis through phosphorylation of elongation factor 2 (eEF-2). It is subject to complex regulation by multiple upstream signaling pathways, through poorly described mechanisms. Precise integration of these signals is critical for eEF-2K to appropriately regulate protein translation rates. Here, an allosteric mechanism comprising two sequential conformations is described for eEF-2K activation. First, Ca2+/CaM binds eEF-2K with high affinity (Kd(CaM) = 24 ± 5 nM) to enhance its ability to autophosphorylate Thr-348 in the regulatory loop (R-loop) by > 104-fold (kauto = 2.6 ± 0.3 s-1). Subsequent binding of phospho-Thr-348 to a conserved basic pocket in the kinase domain potentially drives a conformational transition of the R-loop, which is essential for efficient substrate phosphorylation. Ca2+/CaM binding activates autophosphorylated eEF-2K by allosterically enhancing kcat for peptide substrate phosphorylation by 103-fold. Thr-348 autophosphorylation results in a 25-fold increase in the specificity constant (kcat/Km(Pep-S)), with equal contributions from kcat and Km(Pep-S), suggesting that peptide substrate binding is partly impeded in the unphosphorylated enzyme. In cells, Thr-348 autophosphorylation appears to control the catalytic output of active eEF-2K, contributing more than 5-fold to its ability to promote eEF-2 phosphorylation. Fundamentally, eEF-2K activation appears to be analogous to an amplifier, where output volume may be controlled by either toggling the power switch (switching on the kinase) or altering the volume control (modulating stability of the active R-loop conformation). As upstream signaling events have potential to modulate either allosteric step, this mechanism allows for exquisite control of eEF-2K output.