The molecular mechanism of FbpABC iron(3+) transport in Gram negative pathogenic bacteria

Iron is an essential nutrient required by nearly all forms of life. Pathogenic Haemophilus influenzae, Neisseria spp. (N. gonorrhoeae and N. meningitidis) and other Gram-negative bacteria utilize a periplasm-to-cytosol siderophore-independent FbpABC iron transporter in the acquisition of host-transferrin Fe3+ . The FbpABC transporter is composed of a ferric-ion binding protein (FbpA) and an ABC-transporter consisting of a membrane permease (FbpB) and an ATP-binding protein (FbpC). The association between iron acquisition and human disease suggests that FbpABC may be an important target for antimicrobial drug development. The goal of this dissertation was to develop a detailed model of the molecular mechanism of FbpABC Fe3+ transport. The initial aim involved investigation of the structural basis of hFbpA Fe 3+-binding and release. Through crystallographic and biochemical analyses of Fe3+-free apo-hFbpA, a structural model for the Fe 3+-binding process was devised incorporating information from the previously solved structure of Fe3+-bound holo-hFbpA. This process involves a synergistic anion which binds to apo-hFbpA serving to preorder the binding site prior to Fe3+-loading. Fe3+ coordination proceeds through a kinetically ordered mechanism resulting in a stable Fe3+FbpPO4 ternary complex. The second aim involved development of an experimental model system of hFbpABC transport in Escherichia coli and investigation of the dynamic transport mechanism. Using functional assays, the metal specificity, energy requirements, kinetics of hFbpABC transport and the functional effects of an hFbpA Fe 3+ binding site mutant (hFbpAY196I) were investigated. It was concluded that the specificity and high affinity binding characteristics suggest the transporters function as specialized transporters satisfying the strict chemical requirements of Fe3+ chelation and membrane transport. The final aim involved implementation of functional assays combined with mutagenesis to investigate critical residues within the hFbpB membrane permease. Both site-directed mutagenesis and a novel mutant selection technique based on resistance to the toxic Fe3+ analog Ga3+ were utilized. Computational analysis was used to construct a topological model of the permease and informative residues were mapped to this model. The results of these studies have led to a refined model of the FbpABC Fe3+ transporter and important aspects relative to the molecular mechanism of transport are discussed.