The Haemophilus influenzae hFbpABC Fe3+ Transporter: Analysis of the Membrane Permease and Development of a Gallium-Based Screen for Mutants

Pathogenic bacteria employ a number of acquisition strategies in competition for host iron (Fe3+). Siderophore-dependent iron transport is a widely used strategy that involves the secretion of organic siderophore molecules that compete for iron bound to the high-affinity host transferrin (Tf) and lactoferrin (Lf) proteins (18). Fe3+-siderophore complexes are subsequently recovered by the bacteria through the activity of siderophore-specific surface receptors and transporters. As an alternate strategy, several gram-negative pathogens, including Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenzae, utilize a siderophore-independent (free) Fe3+ transport system (37). In lieu of siderophores, this system employs surface receptors that bind host iron-binding proteins Tf and Lf directly (17, 45). Fe3+ is removed and transported across the outer membrane by the Tf/Lf-binding protein complex (TbpA/TbpB or LbpA/LbpB) using an energy-dependent mechanism mediated by TonB and associated proteins ExbB and ExbD. Naked (free) Fe3+ is transported from the periplasm to the cytosol by the FbpABC transporter, which is composed of a periplasmic ferric ion-binding protein (FbpA) and an inner membrane ABC transporter consisting of a membrane permease (FbpB) and an ATP-binding protein (FbpC) (37). A fundamental difference between siderophore-associated and free iron transport involves the chemical nature of the substrate. In the former, iron is bound and transported into the cytosol as an intact Fe3+-siderophore complex. Coordination of iron in this complex serves a dual purpose of assigning molecular identity to the Fe3+-siderophore for recognition by the appropriate receptors and transport proteins as well as shielding Fe3+ from hydrolysis during transit into the cell (3, 11). In contrast, the free iron transport system lacks any known siderophore; rather, Fe3+ is removed directly from Tf or Lf and transported in free form via direct interaction with specific receptors and Fe3+-binding proteins. These transport proteins must exhibit high specificity and affinity for Fe3+ to avoid insolubility and reactivity, yet they must readily exchange the metal during the process of transport. Our investigations on the homologous FbpABC transporters from H. influenzae hFbpABC (also referred to in the literature as HitABC) and N. gonorrhoeae (nFbpABC) have focused largely on the processes of high-affinity Fe3+ binding and release by the FbpA periplasmic binding proteins. X-ray structures of H. influenzae hFbpA, in both Fe3+-bound (holo) and Fe3+-free (apo) conformations, have provided insight into the Fe3+ coordination complex and the structural transitions involved in substrate binding and release (13, 14). Thermodynamic and kinetic investigations on N. gonorrhoeae nFbpA have shed light on the mechanism of Fe3+ coordination, particularly with respect to the effects of ternary anions and binding site mutations (10, 22-24, 39, 40, 47). It is clear that the FbpA proteins are structural and functional paralogs of the mammalian Tf single iron-binding lobes. In addition to sharing a similar tertiary structure, the FbpA proteins possess a similar set of Fe3+-coordinating residues and undergo a large-scale central hinge rotation upon binding Fe3+ similar to that of Tf. Although Tf and nFbpA demonstrate similar Fe3+ binding affinities (nFbpA, 2.4 × 1018 M−1; N-lobe hTf, 1.8 × 1017 M−1), the proteins exhibit important binding site differences, which may be indicative of dissimilar binding and release mechanisms (47, 51). As evident in the crystal structures of these proteins, hFbpA recruits a monodentate PO4 anion and a water to complete the inner coordination sphere of Fe3+, while Tf (and Lf) enlists a bidentate CO32− anion (6, 9, 14). This distinction may be the result of slightly different protein conformations and a larger, more solvent-exposed FbpA Fe3+-binding site. Importantly, binding site differences correlate to increased exchange and lability of the bound anion and a positive shift in redox potential in nFbpA compared to that of Tf (23, 27, 47). These features have direct influence on the stability of bound Fe3+, with potential implications in the mechanism of transport (10, 22, 47). Recent experiments have demonstrated that the H. influenzae hFbpABC transporter functions as a bona fide binding protein-dependent ABC transporter, employing ATP as an energy source and exhibiting transport rates similar to those of other members of this bacterial ABC transporter family (7). However, the exceptionally high FbpA Fe3+ binding affinity (approximately 1010 to 1012 higher than typical periplasmic binding protein affinities) requires further critical evaluation of FbpABC function and auxiliary processes (anion exchange or redox) that may be involved in the transport process. Clearly, an important event during the transport process is the exchange of Fe3+ from FbpA to the FbpB permease subsequent to transport across the inner membrane. The permease is an ∼500-amino-acid polypeptide proposed to be a polytopic transmembrane protein, forming both a receptor for FbpA and a channel for the passage of Fe3+. The FbpB homologs possess two permease motifs of the template EAA—G———I-LP that are well conserved among the family of bacterial ABC transporter permeases (19, 30, 44). These regions are presumed to reside on cytoplasmically exposed loops that form a mechanical coupling between the energy transduction protein (FbpC) and the membrane transport protein (FbpB). The recent crystal structure of the vitamin B12 ABC transporter (BtuC2D2) verifies a key role for these “L” loop motifs in mediating intimate contact between the permease and ATP binding subunits (34). The hFbpB and nFbpB homologs are highly hydrophobic and toxic when expressed from recombinant sources; thus, despite rigorous isolation efforts, the permease has remained elusive and characterization has been limited to genetic approaches (1). As a logical progression in our studies of the FbpABC system, we have broadened our focus to include the FbpB permease and its role in the Fe3+ transport process. Expression of the H. influenzae hitABC three-gene operon in the siderophore-deficient H-1443 aroB Escherichia coli strain has served as an important model system with which to investigate the function of the hFbpABC transporter (2, 7). In this study, we have utilized this system, coupled with quantitative and qualitative assays, to probe the significance of single amino acids within the hFbpB permease. Multiple-sequence alignments between FbpB permease homologs and related ABC transporter permeases served as a basis for a series of site-directed mutations targeted at residues within the conserved permease motifs. A positive selection screen using the Fe3+ analog gallium (Ga3+) was employed to identify additional mutants, which were genetically delineated and subjected to Fe3+ transport analyses. Finally, a topological model of the hFbpB protein is presented, and implications of informative mutations are discussed in light of a hypothetical functional mechanism of the hFbpB permease protein. These investigations represent an important initial step in probing the structure and function of the heretofore unexplored FbpB permease.