Abstract 2098 The Mechanism of NTP Hydrolysis by Vibrio cholerae NFeoB

In eukaryotes, the elongation factor-2 kinase (eEF2K) downregulates protein biosynthesis by phosphorylating residue T56 on the GTPase eEF2, putatively hindering its ability to engage the ribosome.eEF2K is a member of the atypical family of akinases whose activation, regulation, and catalytic mechanisms remain poorly understood.It is the only a-kinase that relies on calmodulin (CaM) for activation, which occurs through a unique mechanism distinct from other CaM-activated kinases.Upregulation of eEF2K activity involves binding calmodulin (CaM), autophosphorylation on a specific threonine residue (T348), and the intermolecular docking of this phospho-threonine into a conserved regulatory site.Using a combination of biochemical, NMR, HDX-MS, and crystallographic analyses, we provide evidence that the formation of the active configuration is achieved through an allosteric process driven by the C-terminal lobe of CaM that couples to the kinase active site through an "activation spine".We also show that ADP can act as a non-essential activator of eEF2K by stabilizing CaM within the active complex.Upon activation, phosphorylation proceeds through a sequential mechanism where the ATPMg2+ binding precedes substrate loading.ATPMg2+ binding to the active enzyme triggers additional conformational changes, including the closure of the kinase lobes and the appearance of a phosphorylated catalytic aspartate (D284).Our findings shed light on critical steps of the activation process and suggest the potential for a unique phospho-transfer mechanism in a-kinases compared to "typical" kinases.