
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.23.609456
preprint
1
Article
Deep mutational scanning of EccD 3 reveals the molecular basis of its essentiality in the mycobacterium ESX secretion system
Trinidad Donovan D. http://orcid.org/0000-0002-1439-9927

Macdonald Christian B.
Rosenberg Oren S.
Fraser James S. http://orcid.org/0000-0002-5080-2859

Coyote-Maestas Willow http://orcid.org/0000-0001-9614-5340

24 8 2024
2024.08.23.609456https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
http://biorxiv.org/lookup/doi/10.1101/2024.08.23.609456
nihpp-2024.08.23.609456.pdf
Abstract

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 M. tuberculosis survival, is a promising target. In this study, we perform a deep mutational scan on the ESX-3 core protein EccD 3 in the model organism M. 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 EccD 3 . Our data combined with structural comparisons to ESX-5 complexes support a model where EccD 3 stabilizes 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.
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pmc
