PROJECT SUMMARY There is a compelling need to decipher the role of the dysregulation of translation elongation in various chronic neurological conditions and various cancers. The long-term goal driving the proposed research is to help develop therapeutic strategies targeting eEF2K for the clinical treatment of progressive neurodegenerative diseases and malignancies. The overall objectives in this application are to (i) characterize the kinetic mechanism of eEF2K activation, (ii) elucidate the structural basis for the activation and regulation of eEF2K, and (iii) determine a unifying mechanism of the regulation of eEF2K activation and activity by divalent cations, pH, ADP, and post- translational modifications, and (iv) quantify eEF2K-mediated signal transduction in mammalian cells. The central hypothesis is that calmodulin (CaM) binding activates eEF2K by profoundly altering its conformational dynamics, leading to a state capable of efficient phosphoryl transfer. Multiple regulatory inputs control the attainment of this state. The rationale is that understanding the mechanism of eEF2K regulation is necessary to provide a robust scientific framework for developing novel therapeutic approaches targeting neurodegenerative diseases and cancer. The central hypothesis is tested through two specific aims: (1) to determine the mechanistic basis for regulating eEF2K activation and the properties of the active state and (2) to delineate the regulatory influences on eEF2K activation and activity by suppressive elements and post-translational modifications. In the first aim, the precise allosteric mechanism of eEF2K activation will be defined through a combination of pre-steady state kinetics, computational approaches, and structural analysis. The second aim will determine the modulatory effects of specific post-translational modifications in affecting the CaM sensitivity of the active state. The findings for both aims will be validated by characterizing eEF2K activation and activity in mammalian cells. In our opinion, the research proposed in this application is innovative because it focuses on understanding the relationships between the structural dynamics and the temporal control of eEF2K in mammalian cells using a unique combination of techniques integrated over multiple length scales from the atomic to the cellular. The proposed research is significant because it is expected to provide new insight into the cellular regulation of protein translation by eEF2K and provide critical advancement in understanding various eEF2K-driven pathologies.