{"doi":"10.1101/2025.05.13.653565","title":"The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase","abstract":"Abstract Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the α−kinase family, modulates translational rates by phosphorylating eEF-2, a GTPase that facilitates the translocation of the nascent chain on the ribosome during the elongation phase of protein synthesis. eEF-2K is regulated by diverse cellular cues, many of which sensitize it to the Ca 2+ -effector protein calmodulin (CaM). CaM, which binds and allosterically activates eEF-2K in the presence of Ca 2+ , contains two structural “lobes,” each with a pair of Ca 2+ -binding EF-hands. Using kinetic analysis, we demonstrate that the isolated C-terminal lobe of CaM (CaM C ) is sufficient to engage and fully activate eEF-2K in a Ca 2+ -dependent fashion. Genetically fusing CaM C to the N-terminus of eEF-2K, upstream of its critical CaM-targeting motif (CTM) via a flexible 2-glycine linker, results in a chimeric species (C-LiNK) that is constitutively active independent of external CaM and Ca 2+ . A structure of the C-LiNK functional core reveals no significant deviation in the overall conformations of the interacting modules and orientations of key catalytic-site residues relative to the heterodimeric complex between full-length CaM and eEF-2K. These observations demonstrate that, in contrast to other CaM-regulated kinases, CaM C alone is sufficient to activate eEF-2K fully. The proximity effect of CaM C in the context of C-LiNK removes the requirement for external Ca 2+ , whose apparent role is to enhance the CaM-affinity of eEF-2K and drive kinase activation. The responsiveness of eEF-2K to regulatory stimuli in cells appears to be lost in C-LiNK, presumably due to its permanently “on” state.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":557932,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9585,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1068452,"name":"Luke S. Browning","orcid":"0000-0002-1301-321X","position":1,"is_corresponding":false},{"id":751030,"name":"Andrea Piserchio","orcid":"0000-0001-7865-9065","position":2,"is_corresponding":false},{"id":558088,"name":"Eta A. Isiorho","orcid":null,"position":3,"is_corresponding":false},{"id":1241701,"name":"Mohamed I. Gadallah","orcid":"0000-0003-4913-7668","position":4,"is_corresponding":false},{"id":1451333,"name":"Jomai Douangvilay","orcid":null,"position":5,"is_corresponding":false},{"id":104076,"name":"Elizabeth Wang","orcid":"0000-0001-5226-7258","position":6,"is_corresponding":false},{"id":1451334,"name":"Justin K. Kalugin","orcid":null,"position":7,"is_corresponding":false},{"id":215664,"name":"Jennifer S. Brodbelt","orcid":"0000-0003-3207-0217","position":8,"is_corresponding":false},{"id":751032,"name":"Ranajeet Ghose","orcid":"0000-0002-0763-8686","position":9,"is_corresponding":false},{"id":292931,"name":"Kevin N. Dalby","orcid":"0000-0001-9272-5129","position":10,"is_corresponding":false},{"id":751031,"name":"Kimberly J. Long","orcid":"0000-0002-3647-705X","position":0,"is_corresponding":true}],"reference_count":54,"raw_metadata":null,"created_at":"2026-07-19T02:55:21.727661Z","pmid":"40462953","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}