Deep mutational scanning of EccD₃reveals 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 thusM. tuberculosissurvival, is a promising target. In this study, we perform a deep mutational scan on the ESX-3 core protein EccD₃in the model organismM. smegmatis. 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 EccD₃stabilizes the complex, with the hinge motif within the linker being particularly sensitive to disruption. Our study is the first deep mutational scan in mycobacteria, which could help guide drug development toward novel treatment of tuberculosis. This study underscores the importance of context-specific mutational analyses for discovering essential protein interactions within mycobacterial systems.