Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase

Abstract

Translation is a tightly regulated process that ensures optimal protein quality and enables adaptation to energy/nutrient availability. The α-kinase eukaryotic elongation factor 2 kinase (eEF-2K), a key regulator of translation, specifically phosphorylates the guanosine triphosphatase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and autophosphorylation at the primary stimulatory site, T348. Biochemical studies predict a calmodulin-mediated activation mechanism for eEF-2K distinct from other calmodulin-dependent kinases. Here, we resolve the atomic details of this mechanism through a 2.3-Å crystal structure of the heterodimeric complex of calmodulin and the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights an intimate association of the kinase with the calmodulin C-lobe, creating an “activation spine” that connects its amino-terminal calmodulin-targeting motif to its active site through a conserved regulatory element.

eEF2K Inhibitor Design: The Progression of Exemplary Structure-Based Drug Design

“…Crystal structures of MHCKA in complex with AMP-PNP, a non-hydrolysable ATP derivative, and eEF2K in complex with CaM, the latter of which published recently at the time of this writing, supports earlier characterization between TRPM7 and PKA [ 34 , 35 ]. Both MHCKA and eEF2K contain Cys 2—His 2 zinc fingers that coordinate a zinc ion, and an elongated α-helix.…”

“…Though it is worth noting that the crystal structure of eEF2K does not have observable electron density for a ligand in its active site and therefore, side chains may be more flexible [ 35 ]. Homologous residues between eEF2K (blue) and MHCKA (green) are highly conserved within the binding pocket ( Figure 2 ) [ 35 ].…”

“…Though it is worth noting that the crystal structure of eEF2K does not have observable electron density for a ligand in its active site and therefore, side chains may be more flexible [ 35 ]. Homologous residues between eEF2K (blue) and MHCKA (green) are highly conserved within the binding pocket ( Figure 2 ) [ 35 ]. Important MHCKA residues for adenosine nucleotide binding such as E713, Q758, and K645 are conserved in three-dimensional space in the eEF2K crystal structure compared to MHCKA, indicative of a similar binding pocket and likely similar mechanisms.…”

“…Excitingly, in 2022, Piserchio et al deposited a CaM bound eEF2K crystal structure with phosphorylated threonine 348 (PDB:7SHQ) with an accompanying publication [ 35 ]. This undoubtedly will play a significant impact in future eEF2K targeted drug design.…”

“…To date, to the best of our knowledge, no eEF2K inhibitors reported so far have been designed using crystal structure as opposed to a homology model. While the recent structure does not have ATP or a derivative bound in its active site, the deposited crystal structure captures the activated eEF2K enzyme with CaM bound and the autophosphorylation of threonine 348 in its respective pocket [ 35 ]. With existing protein-ligand docking programs, and ATP derivative—MHCKA crystal structures serving as binding pose templates, it is not unreasonable to suggest that the ATP-eEF2K binding pose may be accurately predicted.…”