Phosphorylation of a conserved aspartate in the catalytic site of eukaryotic elongation factor 2 kinase

Eukaryotic elongation factor 2 kinase (eEF-2K) is a member of the α-kinase family of atypical serine/threonine kinases. eEF-2K, the only calmodulin-activated α-kinase, phosphorylates the ribosome-associated GTPase, eukaryotic elongation factor 2 (eEF-2), suppressing translational elongation. α-kinases, including eEF-2K, possess catalytic site geometries that are distinct from those of typical kinases, suggesting possible divergence in their phospho-transfer mechanisms. Unlike typical protein kinases, where chemistry is known to proceed through a sequential mechanism involving a ternary kinase-substrate-ATP•Mg²⁺ complex, the nature of the chemical step catalyzed by α-kinases remains poorly defined. Here, multiple orthogonal lines of evidence, including a crystal structure and solution-state mass spectrometry data, suggest phosphorylation of a catalytically essential aspartate residue (D284) at the eEF-2K active site. Previous crystallographic evidence of the presence of a phospho-aspartate at an equivalent position (D766) in the related Dictyostelium α-kinase MHCK-A strongly suggests that this species represents a conserved active-site feature in α-kinase family members, despite their disparate modes of activation. This observation, together with existing kinetics data on eEF-2K, raises the possibility that phospho-transfer chemistry in α-kinases occurs via an ordered stepwise mechanism involving a phospho-enzyme intermediate, contrasting with typical protein kinases.