Fig. 3: Mutational analysis of mycobactin binding site.

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 middle part of the crevice, Fe-MBT is bound (Dataset #5, PDB 9FW3, OF_occl structure). Residue Q249^rtB interacts with Fe-MBT, and was functionally characterized. b ATPase activity of purified Q249A^IrtB, Q249L^IrtB, Q249F^IrtB, and Q249R^IrtB variants with WT-IrtAB as control in the presence or absence of 5 µM Fe-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 four mycobactin binding site variants. Note that M. smegmatis cells exhibit IrtAB-independent Fe-cMBT uptake. Data points correspond to technical triplicates. a, b 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 Fe-MBT bound 2xEQ-IrtAB (Dataset #5, PDB 9FW3) and the 2xEQ-IrtAB-Q249R^IrtB variant (Dataset #7, PDB 9G2S) unable to bind Fe-MBT owing to crevice collapse.