Bacterial infections pose one of the most significant global public health challenges, exacerbated by the emergence of antibiotic-resistant strains. As a result, treating these infections has become increasingly difficult. Recently, the FDA approved cefiderocol, a siderophore-antibiotic conjugate, as a new treatment for urinary tract infections (UTIs) caused by antibiotic-resistant Gram-negative bacteria. This approval highlights the potential of targeting the bacterial siderophore-mediated metal uptake process for pharmaceutical interventions. A crucial step in this process involves the uptake of metal-chelated siderophores across the bacterial inner membrane, facilitated by ATP-binding cassette (ABC) importers. Unfortunately, the detailed molecular mechanisms underlying these importers remains poorly understood, impeding the development of targeted drugs. This proposal focuses on a unique example of these critical importers: the yersiniabactin importer YbtPQ from uropathogenic E. coli, the leading cause of UTIs. Our preliminary studies have revealed that YbtPQ exhibits folding characteristics similar to an ABC exporter, representing a novel type of ABC transporter. Building upon this discovery, we expect to study the fundamental structure-function relationship of YbtPQ by pursuing two specific aims: 1) Define the substrate selectivity and inhibitory mechanism of YbtPQ; 2) Understand the unique structural motifs within YbtPQ. To accomplish these goals, we will employ a multidisciplinary approach, combining mutagenesis, specifically-designed transport assays, microscale thermophoresis, circular dichroism, native mass spectrometry, molecular dynamic simulations, and cryo-electron microscopy. Successful completion of this proposal will yield valuable insights into the substrate/inhibitor selectivity and transport dynamics of YbtPQ. This knowledge will serve as a solid foundation for future drug development targeting this importer, offering new possibilities for effectively treating bacterial infections and addressing the challenges of antibiotic resistance.