biostudies-literatureUnknownTrinidad DDNIAID NIH HHSNIGMS NIH HHSTuberculosis remains the deadliest infectious disease in the world and requires novel therapeutic targets. The ESX-3 secretion system, which is essential for iron and zinc homeostasis and thus <i>M. tuberculosis</i> survival, is a promising target. In this study, we perform a deep mutational scan on the ESX-3 core protein EccD₃ in the model organism <i>M. smegmatis</i>. We systematically investigated the functional roles of 145 residues across the soluble ubiquitin-like domain, the conformationally distinct flexible linker, and selected transmembrane helices of EccD₃. Our data combined with structural comparisons to ESX-5 complexes support a model where EccD₃ stabilizes the complex, with the hinge motif within the linker being particularly sensitive to disrbioRxiv : the preprint server for biology2024.08.23.609456https://www.ebi.ac.uk/biostudies/studies/S-EPMC11370616biostudies-literatureDeep mutational scanning of EccD₃ reveals the molecular basis of its essentiality in the mycobacterium ESX secretion system.PMC11370616F32 GM152977R35 GM145238F31 AI157438Coyote-Maestas WMacdonald CBTrinidad DDFraser JSRosenberg OSfalseDeep mutational scanning of EccD₃ reveals the molecular basis of its essentiality in the mycobacterium ESX secretion system.Tuberculosis remains the deadliest infectious disease in the world and requires novel therapeutic targets. The ESX-3 secretion system, which is essential for iron and zinc homeostasis and thus <i>M. tuberculosis</i> survival, is a promising target. In this study, we perform a deep mutational scan on the ESX-3 core protein EccD₃ in the model organism <i>M. smegmatis</i>. We systematically investigated the functional roles of 145 residues across the soluble ubiquitin-like domain, the conformationally distinct flexible linker, and selected transmembrane helices of EccD₃. Our data combined with structural comparisons to ESX-5 complexes support a model where EccD₃ stabilizes the complex, with the hinge motif within the linker being particularly sensitive to disr2024-01-01T00:00:00Z2024 Aug2026-04-21T03:28:09.964Z2025-04-06T14:24:14.191ZS-EPMC113706163922917810.1101/2024.08.23.609456