IroT/MavN is a Legionella Transmembrane Fe(II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by In‐vitro Real‐time Transport

Abstract

Iron plays a vital role in all living organisms due to its key role as an enzyme co‐factor, central to diverse metabolic processes. However, because of its high reactivity, speciation, and insolubility in oxidative environments, organisms have evolved sophisticated networks for intercellular iron acquisition without reaching toxic levels. In intravacuolar pathogens, iron is essential for growth and virulence. In Legionella pneumophila, the causative agent of Legionnaires’ disease, a putative transmembrane protein, IroT/MavN, is inserted onto the surface of the host pathogen‐containing vacuole to facilitate intravacuolar iron acquisition from the host, bypassing the problem of Fe(III) insolubility and mobilization. In this work, we have developed a platform for purification and the functional reconstitution of IroT/MavN in artificial lipid bilayer vesicles (proteoliposomes) to identify and characterize the IroT/MavN mediated metal transport properties. By encapsulating the fluorescent reporter probe Fluozin‐3, by real‐time metal transport assays, we revealed that IroT/MavN is a high‐affinity and high‐capacity iron transporter selective for Fe(II) over other essential transition metals. Mutational analysis reveals important residues in the transmembrane helices, soluble domains and loops important for substrate recognition and rapid‐kinetic translocation. In addition, by encapsulating the pH indicator pyranine, we demonstrated that Fe(II) translocation is coupled to H+ counter‐transport, suggesting that IroT/MavN is a Fe(II)‐H+ antiporter. This work establishes the substrate transport properties involved in a novel transporter family important for iron acquisition at the host‐pathogen intravacuolar interface and provides chemical tools for comparative investigation of the translocation properties in other iron transporter families. Support or Funding Information The work was supported by the Robert A. Welch Foundation (AT‐1935‐20170325 to G.M.) and by the National Institute of General Medical Sciences of the National Institutes of Health (R35GM128704 to G.M.).

Stabilization of Supramolecular Membrane Protein-Lipid Bilayer Assemblies Through Immobilization in a Crystalline Exoskeleton

“…47 IroT topology is characterized by 8 transmembrane (TM) helices and a long C-terminal domain. 48,49 The structure and substrate translocation modality in IroT are active areas of research, but much has been gleaned from reconstituting IroT in artificial lipid bilayer systems and performing real-time transport assays. 49 IroT was shown to act as a Fe 2+ /H + antiporter that allows Fe 2+ acquisition into the vacuole from the host cell for pathogen survival.…”

“…48,49 The structure and substrate translocation modality in IroT are active areas of research, but much has been gleaned from reconstituting IroT in artificial lipid bilayer systems and performing real-time transport assays. 49 IroT was shown to act as a Fe 2+ /H + antiporter that allows Fe 2+ acquisition into the vacuole from the host cell for pathogen survival. 48 The second protein selected is a copper P1B-type ATPase from E. coli (CopA), a transmembrane primary-active pump, and part of the P-type ATPase superfamily, that utilize energy generated by ATP hydrolysis to drive Cu + transport across biological membranes against electrochemical gradients.…”

Ins and Outs: Recent Advancements in Membrane Protein-Mediated Prokaryotic Ferrous Iron Transport

“…To meet the iron requirements of the L. pneumophila, IroT is recruited to the LCV membrane. 66,67 IroT is predicted to contain eight transmembrane α-helices and has also been shown to transport a wide array of divalent metals in addition to Fe 2+ , including Mn 2+ , Co 2+ , and Zn 2+ , with equivalent proficiency at least in vitro; however, IroT is suggested to function only for Fe 2+ transport in vivo. 68 While the exact mechanism of IroTmediated metal transport has not been elucidated, several mutagenesis experiments display the importance of key amino acids for metal translocation.…”

Stabilization of Supramolecular Membrane Protein-Lipid Bilayer Assemblies Through Immobilization in a Crystalline Exoskeleton

“…42 IroT topology is characterized by 8 transmembrane (TM) helices and a long C-terminal domain. 43,44 The structure and substrate translocation modality in IroT are active areas of research, but much has been gleaned from reconstituting IroT in artificial lipid bilayer systems and performing real-time transport assays. 44 IroT was shown to act as a Fe 2+ /H + antiporter that allows Fe 2+ acquisition into the vacuole from the host cell for pathogen survival.…”

“…43,44 The structure and substrate translocation modality in IroT are active areas of research, but much has been gleaned from reconstituting IroT in artificial lipid bilayer systems and performing real-time transport assays. 44 IroT was shown to act as a Fe 2+ /H + antiporter that allows Fe 2+ acquisition into the vacuole from the host cell for pathogen survival. 43 The second protein selected is a copper P1B-type ATPase from E. coli (CopA), a transmembrane primary-active pump, and part of the P-type ATPase superfamily, that utilize energy generated by ATP hydrolysis to drive Cu + transport across biological membranes against electrochemical gradients.…”