Periodic Reporting for period 4 - MycoRailway (Discovery and molecular investigation of mycobacterial transporters responsible for iron acquisition)
Reporting period: 2022-10-01 to 2024-03-31
Mycobacterium tuberculosis is a devastating human pathogen, which killed 1.4 million people in 2022. In order to survive and replicate in the phagosome of macrophages where iron levels are kept extremely low, M. tuberculosis relies on the biosynthesis of siderophores called mycobactins, which capture iron with high affinity inside the host cell. Despite their undisputed importance for virulence, little was known at the onset of this project about how these siderophores are exported and imported across the two mycobacterial membranes. The aim of this project was to unravel the structure and mechanism of transporter proteins sitting in the inner and outer mycobacterial membrane involved in the export of empty and the import of iron-loaded mycobactins.
Access to iron represents an Achilles heel of M. tuberculosis and basic research that is conducted in this project to understand iron acquisition at the molecular likely will help to generate new generations of urgently needed drugs against this devastating pathogen.
Access to iron represents an Achilles heel of M. tuberculosis and basic research that is conducted in this project to understand iron acquisition at the molecular likely will help to generate new generations of urgently needed drugs against this devastating pathogen.
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.
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.
This project bridged the disciplines of structural biology (in particular cryo-EM) with protein biochemistry and microbiology using a unique blend of techniques.
Despite the fact that M. tuberculosis is one of the most extensively studied organisms, its complex cell wall as well as technical hurdles to purify and characterize mycobacterial membrane proteins has so far impeded the molecular investigation of siderophore transport. On the other hand, a number of excellent pioneering studies have shed light on genetic and functional aspects of mycobactin-mediated iron acquisition, upon which experimental work of this proposal built on. The project delivered unprecedented insights into proteins responsible for siderophore transport across the inner mycobacterial membranes. As a whole, the project shed light on the molecular mechanisms of mycobacterial membrane transport, which is a terra incognita and still awaits fascinating scientific discoveries. At the translational level, our research facilitates the development of novel cures to treat globally emerging multidrug resistant strains of M. tuberculosis.
Despite the fact that M. tuberculosis is one of the most extensively studied organisms, its complex cell wall as well as technical hurdles to purify and characterize mycobacterial membrane proteins has so far impeded the molecular investigation of siderophore transport. On the other hand, a number of excellent pioneering studies have shed light on genetic and functional aspects of mycobactin-mediated iron acquisition, upon which experimental work of this proposal built on. The project delivered unprecedented insights into proteins responsible for siderophore transport across the inner mycobacterial membranes. As a whole, the project shed light on the molecular mechanisms of mycobacterial membrane transport, which is a terra incognita and still awaits fascinating scientific discoveries. At the translational level, our research facilitates the development of novel cures to treat globally emerging multidrug resistant strains of M. tuberculosis.