Abstract 1911 Calmodulin-mediated activation of elongation factor 2 kinase: insights into the roles of specific residues and domains

A range of cellular processes, including memory consolidation, energy management, and protein folding, rely on a precisely controlled elongation rate during protein synthesis. Dysregulated protein elongation is associated with tumorigenesis and neurodegenerative diseases such as Alzheimer's and Parkinson's. Eukaryotic elongation factor 2 kinase (eEF-2K) plays a crucial role in regulating elongation rates through its inhibitory phosphorylation of eEF-2, an essential component in eukaryotic protein elongation. Various cellular signals tune the activity of eEF-2K, and it is hypothesized that all regulatory inputs converge on a central point of control: modulation of the interaction between eEF-2K and its essential activator protein, calmodulin–a calcium sensor protein. eEF-2K is the only known Ca2+-calmodulin-dependent member of the atypical-kinase family, a rare group distinguished by sequences and domain organizations dissimilar to typical kinases, making the interaction between eEF-2K and calmodulin unique. Despite the exceptional nature of this interaction, the mechanism by which calmodulin binding leads to conformational activation of the enzyme is not well understood. Here, we explore essential components at the calmodulin-eEF-2K interface to elucidate the distinctive mechanism underlying calmodulin-mediated activation of eEF-2K. We hypothesize that the activation of eEF-2K by calmodulin is powered by the binding of calmodulin's C-terminal lobe to the calmodulin-targeting motif. The C-terminal lobe then collapses to make additional contacts with the kinase domain, driving the formation of an activated state. Using stopped-flow, we measured the binding rate between eEF-2K and a fluorescent Ca2+-calmodulin and found a rapid association rate (108 M-1s-1). This suggests that the initial rapid binding step limits the formation of the complex, and conformational activation exceeds the diffusion rate. Then, guided by structural information, we probed specific residues and domains for their binding and activation roles. We created mutations within the activator-enzyme and enzyme-enzyme interface that we predicted are crucial for activation (by calmodulin or intrinsically), and we tested our predictions through kinetic analysis. Our findings demonstrate that intramolecular interactions between the eEF-2K regulatory element and kinase domain facilitate the intrinsic activity of the enzyme, and allosteric contacts between calmodulin and eEF-2K stabilize this activated state. Using a truncated construct of calmodulin, we investigated the role of calmodulin's C-terminal lobe in eEF-2K binding, activation, and calcium sensitivity. Our data revealed no difference in binding or activation by the C-terminal lobe construct compared to wild-type calmodulin. Additionally, we discovered that the C-terminal lobe contains the necessary core for eEF-2K calcium sensitivity. Our data suggest that calmodulin rapidly binds eEF-2K, and calmodulin's C-terminal lobe activates eEF-2K by stabilizing intramolecular contacts necessary for efficient catalysis. We applied the information from these studies and previous crystallographic data to create a constitutively active fusion protein. The novel calmodulin-eEF-2K fusion protein is an investigative tool that can be used to examine regulatory mechanisms and consequences of suppressed elongation rates. This work is supported by NIH award R01 GM123252.