Structural Basis of Gating by the Outer Membrane Transporter FecA

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Science  01 Mar 2002:
Vol. 295, Issue 5560, pp. 1715-1719
DOI: 10.1126/science.1067313


Siderophore-mediated acquisition systems facilitate iron uptake. We present the crystallographic structure of the integral outer membrane receptor FecA from Escherichia coli with and without ferric citrate at 2.5 and 2.0 angstrom resolution. FecA is composed of three distinct domains: the barrel, plug, and NH2-terminal extension. Binding of ferric citrate triggers a conformational change of the extracellular loops that close the external pocket of FecA. Ligand-induced allosteric transitions are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. These data establish the structural basis of gating for receptors dependent on the cytoplasmic membrane protein TonB. By compiling available data for this family of receptors, we propose a mechanism for the energy-dependent transport of siderophores.

Despite its relative abundance in Earth's crust, iron is biologically unavailable in an oxidizing atmosphere because of the insolubility of ferric oxyhydroxide. In response to iron deficiency, most microbes secrete organic chelators called siderophores, which are designed to sequester ferric iron. The ability to acquire this metal is an important determinant of bacterial virulence. Most bacteria express a sophisticated repertoire of parallel iron acquisition systems (1), which underscores their biological importance and the clinical potential to exploit these pathways for combating multi–drug-resistant bacterial strains (2).

Regulatory mechanisms, responsive to both the internal and external iron concentration, control the transcription of genes involved in iron uptake (3). The ferric citrate uptake (fec) genes are responsible for the transport of ferric citrate from the external medium into the cytoplasm (4–7). Proteins required for each phase of these energy-dependent transport processes have defined functions and are localized to specific cell envelope compartments. Embedded within the outer membrane is FecA, which performs two mutually independent functions: It binds and transports ferric citrate, and it is required to initiate transcription of the fecABCDEtransport operon but not the regulatory fecIR genes (4–7). Both siderophore transport and the initiation of transcription require the chemiosmotic potential of the cytoplasmic membrane as transduced by a complex composed of the proteins TonB, ExbB, and ExbD. However, transport is not required for the initiation of transcription; rather, siderophore binding to FecA initiates a signaling cascade from the cell surface, resulting in transcription in the cytoplasm (4–7). The transmembrane regulatory protein FecR activates the cytoplasmic sigma factor FecI to bind to thefecA promoter (4–7). We therefore sought to probe the molecular architecture of FecA and its role in siderophore-induced transcription.

The three-dimensional structures of unliganded FecA and its complex with dinuclear ferric citrate were determined at 2.0 and 2.5 Å resolution by x-ray crystallography using multiple anomalous dispersion (MAD) (Table 1) (8,9). The crystal structure of full-length FecA (741 residues) is composed of three domains (Fig. 1, A and B) and has an overall topology similar to those of FhuA (9–11) and FepA (9, 12). A monomeric 22-stranded β barrel is formed by residues 222 to 741. In vivo, these antiparallel strands traverse the outer membrane. Adjacent strands are connected by solvent-accessible extracellular loops containing two α helices and three β strands; turns are oriented toward the periplasm. The position and length of these connecting segments are different for each receptor (9). When viewed along its axis, the barrel has ellipsoidal dimensions (35 Å by 47 Å) and is 65 Å in height, extending 30 Å above the external interface of the upper leaflet of the outer membrane. Two girdles of aromatic residues inscribe the membrane-embedded hydrophobic surface of the receptor.

Figure 1

Crystallographic structure of FecA, (A) unliganded and (B) liganded. The 22-stranded β barrel is shown in blue. The molecule is presented as found in the outer membrane with extracellular loops extending into the solvent (top) and periplasmic turns oriented toward the periplasm (bottom). Extracellular loops 7 and 8, which undergo major conformational changes upon ligand binding, are shown in red. The mixed four-stranded β sheet of the plug domain is shown in green, with helices in purple and loops in yellow. The switch helix, located in the periplasmic pocket of FecA, is colored orange and is only observed in the unliganded conformation. Dinuclear ferric citrate is represented as a bond model with oxygen atoms in red and carbon atoms in green; both ferric ions are shown as orange spheres. (C) Surface representation of unliganded FecA. Although ferric citrate is not found in the unliganded structure, it is represented as a bond model (with carbon atoms in green, oxygen atoms in red, and ferric ions in orange) to indicate the location of the binding site. (D) Surface representation of liganded FecA. The binding of ferric citrate causes conformational changes of the extracellular loops, such that the siderophore becomes solvent-inaccessible. The molecular surfaces are colored according to electrostatic potential, with blue and red corresponding to +40 kT and –40 kT, respectively. The view is given from the solvent. All figures were prepared using BobScript (42), GLR (43), POV-Ray, and GRASP (44).

Table 1

Data collection, phasing, and refinement statistics.

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The plug domain (residues 80 to 221) is located inside the barrel, comprising five helices, two β strands, and a mixed four-stranded β sheet that is tilted by about 45° with respect to the membrane plane (Fig. 1, A and B). FhuA and FepA present a similar fold (9). The plug domain prevents the direct passage of ferric citrate across the outer membrane and separates the external and periplasmic pockets, which are located above and below the four-stranded β sheet. The extracellular pocket of FecA is lined with positively charged residues (ferric citrate is negatively charged). The equivalent regions of FhuA and FepA are predominantly lined with aromatic and/or hydrophilic residues (10–12). The electrostatic properties of these pockets confer specificity on the basis of the chemical attributes of the siderophore. The flexible 79-residue domain of FecA termed the NH2-terminal extension (residues 1 to 79), which resides entirely within the periplasm and transmits the liganded status of the receptor to FecR (4–7), was not visible in the electron density maps and could not be modeled.

Noncovalently bound within the external pocket of FecA is a single dinuclear ferric citrate molecule (13). Three loops of the plug domain—apices A (Thr138), B (Arg155), and C (Ser180)—extend above the plane of the upper leaflet of the outer membrane. In the liganded structure, apices A, B, and C, together with residues located on extracellular loops 4, 5, 8, and 11, form hydrogen bonds and electrostatic interactions with dinuclear ferric citrate (Fig. 2). Additional van der Waals contacts are provided by Leu156 from the plug domain and Phe333 from the barrel. The binding site of FhuA is markedly different from the binding site of FecA. Although apices A, B, and C of FhuA and several barrel residues also form specific interactions with the ligand, the binding site of FhuA is predominantly formed by hydrophobic residues (10–12, 14, 15).

Figure 2

Stereoview of the ferric citrate-binding site. All side chains within 3.5 Å of dinuclear ferric dicitrate are shown with carbon atoms in white, nitrogen atoms in blue, and oxygen atoms in red. Several water molecules found within the binding site mediate additional protein-ligand interactions. The strands and extracellular loops of the barrel are shown in blue; the strands forming the plug domain are in green, and loops are in yellow. The seventh, eighth, and eleventh extracelluar loops of the barrel are shown in red. The dinuclear ferric citrate molecule is represented as a bond model with oxygen atoms in red, carbon atoms in green, and ferric ions in orange.

Formation of the liganded complex substantially affects the conformation of the barrel and the plug domain of FecA. Binding of ferric citrate causes both minor and major changes in the spatial arrangement and conformation of five extracellular loops (Fig. 1, A and B). Minor changes (<0.5 Å) are observed in the fourth, fifth, and ninth extracellular loops. Strikingly, the seventh extracellular loop (residues 516 to 535) is translated by up to 11 Å, and it changes conformation such that helix 2 (residues 522 to 529) is unwound. Residues 562 to 581 from the eighth extracellular loop are also translated by up to 15 Å.

Allosteric transitions tightly regulate the opening and closing of TonB-dependent receptor channels, where these events are modulated by (i) siderophore binding and (ii) the transfer of stored potential energy from TonB. This bipartite gating mechanism refers to the process by which siderophore binding to its receptor induces allosteric transitions that enable transport by establishing or removing a physical channel obstruction, referred to as the gate (16). Opening and closing of the two gates regulates transmembrane siderophore flow. Diverse organisms, from bacteria to humans, use gating mechanisms in the regulated influx and efflux of solutes across biological membranes. The rearrangement of the seventh and eighth extracellular loops of FecA establishes the structural basis of gating for TonB-dependent receptors. In FecA, these movements render the external pocket and the ferric citrate-binding site inaccessible to solvent. Hence, the barrel of a TonB-dependent receptor is a dynamic entity that actively participates in the energy-dependent siderophore uptake (Fig. 1, C and D).

Allosteric transitions are propagated across the outer membrane by the plug domain via shifts (0.5 to 0.7 Å) toward the ferric citrate molecule of apices A and C, and of apex B away from the siderophore, and via the concerted downward movement (0.7 to 2.0 Å) toward the periplasm of βE and βF (10, 11, 17). In the liganded structure, an NH2-terminal segment located within the periplasmic pocket of FecA, termed the switch helix (10), unwinds to assume a flexible extended conformation (as judged by the absence of interpretable electron density before residue Asn95). Similar changes have been observed in the three-dimensional structures of multiple liganded complexes of FhuA (10, 11, 14, 15). The unwinding of the switch helix signals the occupancy of the receptor in the periplasm such that energized TonB molecules can effectively discriminate between unliganded and liganded receptors (18).

The TonB-box is a functionally important region of TonB-dependent receptors (19) that physically interacts with TonB and is essential for the transfer of the chemiosmotic potential of the cytoplasmic membrane as transduced by the TonB-ExbB-ExbD complex (20–23). After ferric citrate binding, the TonB box of FecA (residues 80 to 84) becomes disordered, as evidenced by its lack of discernible electron density in the liganded structure.

By consolidating these data with other available genetic, biochemical, and structural observations for this family of TonB-dependent receptors, we have derived the following transport mechanism.

Stage 1: The siderophore is adsorbed from the medium with low affinity, primarily by aromatic residues found within the upper portion of the external pocket of the unliganded receptor.

Stage 2: The siderophore is transferred to its high-affinity binding site, where primarily apices A, B, and C of the plug domain and numerous charged residues found within the extracellular loops and β strands of the barrel form electrostatic contacts with the siderophore. These interactions cause translations of apices A, B, and C toward and away from the siderophore, as well as the concerted downward movement of other plug domain segments, leading to the unwinding and repositioning of the switch helix and the TonB-box.

Stage 3: Multiple extracellular loops of the receptor change their relative conformation and position. This bipartite gating mechanism closes the external pocket of the barrel, thereby favoring directed transport by disrupting the low-affinity binding site and shielding the high-affinity binding site from the solvent. However, the structural transitions observed in stages 2 and 3 may also be concerted.

Stage 4: Given the absence of an unobstructed channel of sufficient size to accommodate the passage of siderophores through TonB-dependent receptors, transitions that modify the conformation of the plug domain and/or barrel are required for transport. These changes are mediated by physical interactions between TonB and the TonB-box (20–23), and possibly by periplasmic turns of the barrel (24). Formation of a complex with TonB results in the transfer of stored potential energy to the receptor (25), which in turn promotes ligand transport.

Although the structural basis of the final energy-dependent transport stage remains to be established, two plausible models have been proposed (10–12). In the first proposal, an energized TonB molecule induces allosteric transitions within the plug domain and/or barrel, such that the unfolded plug domain with bound ligand is ejected into the periplasm. In the second proposal, the plug domain remains inside the barrel, and both domains undergo allosteric transitions that lead to the opening of an underlying transmembrane channel within the receptor, through which the siderophore permeates into the periplasm by a surface-diffusion mechanism (similar to those characterized for glycoporins). Biophysical studies recently revealed that in the absence of the FepA barrel, the plug domain is predominantly unfolded yet soluble and retains some binding affinity (26).

  • * To whom correspondence should be addressed. E-mail: johann.deisenhofer{at}


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