Covalent Chemical Tagging of Transmembrane Transport Proteins Illuminates the Internalization Pathways of Xenosiderophores

Siderophore-mediated, transmembrane uptake of iron in bacteria is a complex mechanism of essential nutrient acquisition. In addition to transporters that specifically recognize and shuttle endogenous siderophores, bacteria possess transmembrane transporters that efficiently internalize siderophore-metal complexes produced by other organisms. The discovery of transmembrane transporters using indirect methods has enabled the identification of high-affinity transporters but limits access to lower-affinity, promiscuous transport systems. Therefore, covalent tagging strategies that enable the direct identification of target proteins are desirable. To this end, we examined the co-crystal structure of Fe-D1, a ciprofloxacin-linked ferrioxamine complex bound to thePseudomonas aeruginosa outer-membrane transporter FoxA with affinity for ferrioxamine B and E. Using a rational design approach, we identified ideal structural characteristics for ferrioxamine-based, covalent photo-cross-linker probes with reactivity toward tyrosine- and aspartate-rich binding sites within transporter domains. Fe and Ga complexes of DFO-azir-05 and DFO-azir-06 efficiently tag FoxA inEscherichia coli Lemo21 (DE3) mutants overexpressing FoxA. Subsequently, DFO-azir-06 was successfully used to directly tag and identify the ferrioxamine-binding proteins FoxA and FpvB inP. aeruginosa PAO1, as well as the two main transporters of ferrioxamine B inE. coli K-12, FhuA and FhuE. Moreover, DFO-azir-06 revealed a putative new role of cobalamine transporter BtuB in the transport of DFO derivatives. In conclusion, we demonstrate that careful structural design of covalent, photo-cross-linking siderophore conjugates can provide unprecedented access to the elucidation of siderophore-mediated metal ion uptake in bacteria.