As one of the main highlights, we published a paper on the IrtAB transporter in Nature. IrtAB is an unusual ABC exporter, which plays a major role in the uptake of iron-loaded siderophores in mycobacteria. In addition, it contains a cytoplasmic siderophore interacting domain (SID) responsible for iron reduction and intracellular release. We obtained a 6.7 Å cryo-EM structure of full-length IrtAB and in addition solved high resolution crystal structures of the SID (1.7 Å) and the transporter devoid of the SID (2.7 Å). Together with biochemical and functional in vivo experiments, we were able to show that IrtAB indeed is a siderophore importer. The structure suggested that the SID is placed adequately to reduce membrane bound siderophores, an unexpected finding that was further supported by in vitro experiments. In a paper that is under revision at Nature Communications, we report on a cryo-EM structure of outward-occluded IrtAB in complex with its natural substrate mycobactin. Using site-directed mutagenesis, we identified a highly conserved histidine triad that is important for coupling transport to the ATPase activity of IrtAB. By employing double electron-electron resonance, we could show that IrtAB alternates between the outward-occluded and the inward-facing conformation and does not adopt an outward-facing conformation. These insights were of crucial relevance to formulate a novel transport mechanism that explains the molecular determinants of mycobactin influx mediated by an ABC transporter adopting an ABC exporter fold.
With regards to the siderophore exporters MmpL4 and MmpL5, we succeeded to determine a 3.1 Å cryo-EM structure of MmpL4 in complex with its natural substrate, desferrated mycobactin. In a series of functional experiments conducted with our collaborator Dr. Michael Niederweis (US), we could prove the functional importance of the novel mycobactin binding site for the export of mycobactins, but also the important reserve TB drug bedaquiline, which is effluxed by MmpL4 and MmpL5.
Further, we determined a combined X-ray/cryo-EM structure of the triacylglyceride exporter Rv1410, which works hand-in-hand with the lipoprotein LprG. The corresponding work published in Nature Communications describes molecular hallmarks of Rv1410 making it perfectly equipped to export apolar triacylglycerides.
One aim of our proposed work was to identify novel siderophore transporter components by Tn-seq. Unfortunately, we were scooped in this regards and changed our plans to study PE/PPE proteins, which have emerged as a poorly studied yet important class of proteins likely acting as porins in M. tuberculosis. Within this novel PE/PPE project, we have engineered a Mycobacterium smegmatis strains that produce elevated levels of PE/PPE proteins and continue to explore the uncharted territory of the mycobacterial outer membrane, called the mycomembrane.
