Fig. 4: Mutational analysis of the membrane-facing crevice.

a The membrane-facing crevice opens up between TMH2^IrtA and TMH5^IrtB in the OF_occl conformation. IrtA is colored teal, and IrtB is magenta. In the upper part of the crevice, a hydrophobic pocket is formed, which is occupied with a lipidic moiety (Dataset #5, PDB 9FW3, OF_occl structure). Residue A256^IrtB, located below the lipid-binding pocket, is shown as a green stick and was functionally characterized. b ATPase activity of purified A256L^IrtB, A256F^IrtB, and A256R^IrtB variants with WT-IrtAB as control in the presence or absence of 5 µM MBT. Data points correspond to technical triplicates. c In vivo uptake of ⁵⁵Fe-cMBT in M. smegmatis cells unable to produce their own siderophores and lacking the genomic copy of irtAB. Cells were complemented with WT-IrtAB (positive control), ATPase-deficient 2xEQ-IrtAB (negative control), and the three lateral crevice variants. Note that M. smegmatis cells exhibit IrtAB-independent Fe-cMBT uptake. Data points correspond to technical triplicates. b, c For both functional assays, representative data of at least two biological replicates are shown. Source data are provided as a Source Data file. d Structural comparison of 2xEQ-IrtAB with bound Fe-MBT (Dataset #5, PDB 9FW3) and 2xEQ-IrtAB_ A256R^IrtB (Dataset #8, PDB 9G2T, Arg256^IrtB pointing to protein inside) unable to bind Fe-MBT and exhibiting a collapsed membrane-facing crevice.