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Project summary: (Original summary of the funded parent project R35 GM146862: Molecular Mechanism of Translation Regulation by the eEF2K pathway) This research project aims to provide a mechanistic and structural model of general and location- dependent eukaryotic elongation factor 2 kinase (eEF2K) regulation and its downstream effects on the ribosome. eEF2K is at the confluence of multiple upstream pathways whose signals it integrates. eEF2K’s only known target is the eukaryotic elongation factor 2 (eEF2), which it phosphorylates on a single site. This eEF2K/eEF2- axis is the predominant regulator of translation elongation and has a general role in cell homeostasis. It is also an essential cue-dependent regulator of protein synthesis in localized regions of specific cell types, for example, after neurotransmitter exposure in the synapses of neurons. eEF2 promotes the translation of specific mRNAs while generally inhibiting translation. The mechanism of this paradoxical phenomenon is entirely unknown. We recently showed that active eEF2K imposes a general translation shutdown in which phosphorylated eEF2 and the phase-separating protein SERBP1 stably bind to ribosomes and renders them idle. This assembly suggests possible mechanisms of eEF2-phosphorylation with respect to ribosome stability, mRNA decay, and ribosome localization that collectively explain how eEF2-phosphorylation might lead to preferential translation of certain mRNAs. eEF2K is associated with numerous human diseases, including neurological dysfunctions, infectious diseases, cancers, and autoimmune disorders. Therapeutics targeting eEF2K are under development but currently lack insufficient specificity. Thus, a mechanistic understanding of eEF2K-regulation and its downstream effects are needed. Under this award, we will pursue two key directions: 1) determine how eEF2K structurally integrates signals from upstream pathways and affects its downstream regulation of eEF2, and 2) determine how phosphorylated eEF2 and idle ribosomes regulate mRNA translation globally and locally. Here, we will test the novel hypotheses that eEF2-phosphorylation regulates ribosome stability, mRNA stability, and ribosome localization, which collectively confers the preferential translation of a specific mRNA subset. We will use an integrated structural biology approach using single-particle and in situ cryogenic electron microscopy, paired with biophysical, biochemical, and cell biology approaches to address these general and location-specific roles of the eukaryotic elongation factor 2 kinase (eEF2K) pathway. Our proposed research program will open the door to promising therapeutic approaches for the long list of eEF2K-related human diseases and, more broadly, expand our understanding of translation elongation.