Exchangeability of N Termini in the Ligand-gated Porins ofEscherichia coli

Abstract

The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin ␤-barrel formed by their C termini. We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively. The resultant chimeric proteins and empty ␤-barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses. Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane ␤-barrel domain. Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound. The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type. The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V. (1999) Mol. Microbiol. 33, 1037-1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization.

Dimerization of TonB Is Not Essential for Its Binding to the Outer Membrane Siderophore Receptor FhuA of Escherichia coli

“…Furthermore, the globular domains of FhuA and FepA are exchangeable without loss of substrate specificity. For example, a mixed mutant consisting of an FhuA-barrel and an FepA-cork retains the specificity for ferrichrome, the natural substrate for FhuA (23). Different cork-barrel combinations from several bacterial strains led to the same results (24).…”

Differential substrate-induced signaling through the TonB-dependent transporter BtuB

“…TonB may also interact with other parts of the transporters, as implied from genetic suppression patterns (15). Claims that FepA and FhuA variants lacking the hatch domain and Ton box (32,33) still exhibit TonB-dependent activity have been challenged by the recognition that the empty barrels can be complemented by the hatch domains encoded by the mutated chromosomal alleles (34). The N-terminal regions of FhuA and FecA move extensively after substrate binding, but the Ton box regions themselves are disordered.…”

Siderophore Transport through Escherichia coli Outer Membrane Receptor FhuA with Disulfide-tethered Cork and Barrel Domains

“…Ferric siderophore transport requires a pathway with a minimal diameter of ϳ15 Å, which is not apparent in either apo-FhuA (Protein Data Bank code 2FCP) or the ferrichrome-bound crystal structure (Protein Data Bank code 1FCP) (1,4). Comparison of these two FhuA structures revealed distinct conformations; most differences are localized to the cork domain, thereby providing direct evidence for conformational changes that occur within FhuA upon ligand binding.…”

“…Deletions of the cork domains from the outer membrane receptors FhuA and FecA result in decreased affinity for siderophores, but retention of TonB-dependent functions (4,12). It has been postulated that displacement of the siderophore from its initial binding site drives an inward motion of the extracellular loops, resulting in removal of the cork from the barrel lumen (4). In this model, the siderophore could pass through a transiently formed pathway by diffusion.…”

“…However, recent molecular dynamics simulations suggest that solvation of polar interactions within this network can reduce the energy expenditure necessary for cork removal (15). Deletions of the cork domains from the outer membrane receptors FhuA and FecA result in decreased affinity for siderophores, but retention of TonB-dependent functions (4,12). It has been postulated that displacement of the siderophore from its initial binding site drives an inward motion of the extracellular loops, resulting in removal of the cork from the barrel lumen (4).…”