Deep mutational scanning of M. smegmatis EccD3

M. smegmatis and pathogenic mycobacteria iron acquisition is mediated by the ESX-3 secretion system. We lack an understanding of how ESX-3 secretion occurs, and what molecular motions underlie this function. We studied the ESX-3 complex core protein EccD3, which is central within the complex and makes contacts with each of the other core proteins. Comprehensive mapping of all possible mutations at each position in EccD3 would enable determination of which residues are required for ESX-3 function. This information will be critical to determine which intracomplex interactions are important for function. Here, we perform a deep mutational scan of the EccD3 ubiquitin-like domain, the linker connected this domain to the transmembrane domain, and select residues within the transmembrane domain. With this approach, we have identified residues required for EccD3 stability, a network of key interactions between the linker and other core proteins, and notable interactions in the transmembrane helices.