Structure of TonB in Complex with FhuA, E. coli Outer Membrane Receptor

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Science  02 Jun 2006:
Vol. 312, Issue 5778, pp. 1399-1402
DOI: 10.1126/science.1128057


The cytoplasmic membrane protein TonB spans the periplasm of the Gram-negative bacterial cell envelope, contacts cognate outer membrane receptors, and facilitates siderophore transport. The outer membrane receptor FhuA from Escherichia coli mediates TonB-dependent import of ferrichrome. We report the 3.3 angstrom resolution crystal structure of the TonB carboxyl-terminal domain in complex with FhuA. TonB contacts stabilize FhuA's amino-terminal residues, including those of the consensus Ton box sequence that form an interprotein β sheet with TonB through strand exchange. The highly conserved TonB residue arginine-166 is oriented to form multiple contacts with the FhuA cork, the globular domain enclosed by the β barrel.

Iron is an essential element in bacteria for a number of redox processes (1). It is highly insoluble in its ferric (Fe3+) form at physiological pH. To overcome the low bioavailability of Fe3+, Gram-negative bacteria have evolved strategies for high-affinity Fe3+ uptake. One well-characterized strategy involves transduction of energy from the cytoplasmic membrane (CM) to the outer membrane (OM), resulting in active transport of Fe3+ chelated to molecules known as siderophores. Central to this process, the CM-anchored energy-transducing protein TonB spans the periplasm and contacts OM-embedded receptors that bind metal-chelated siderophores. TonB, through uncharacterized conformational changes, transduces energy from the CM's proton motive force to the receptor and facilitates siderophore transport into the periplasm. Existing structural data that describe key protein components of the siderophore transport cycle highlight outstanding questions regarding the overall molecular mechanism (2).

The CM protein TonB is composed of three domains. An N-terminal domain (residues 1 to 33) is anchored to the CM and makes contacts with proteins ExbB and ExbD to form an energy-transducing complex. A C-terminal domain (residues 155 to 239) directly contacts receptors in the OM. An intermediate domain (residues 34 to 154) contains a region of alternating Pro-Glu and Pro-Lys repeats located between residues 66 and 100. There is currently no known structure for the entire TonB protein. However, three structures have been reported for the TonB C-terminal domain. Its oligomeric states are variable and correlate with lengths of the recombinant constructs used to obtain the structures: a tightly interwoven dimer, residues 155 to 239 (3); a loose dimer, residues 148 to 239 (4); and a monomer, residues 103 to 239 (5). The monomeric C-terminal domain consists of two α helices positioned on the same face of a four-stranded antiparallel β sheet, strand β4 being located at the C terminus of TonB.

The OM receptor FhuA is the Escherichia coli transporter of the hydroxamate siderophore ferrichrome. Structurally homologous TonB-dependent OM receptors include the E. coli transporters of cobalamin (BtuB) (6), ferric citrate (FecA) (7), and ferric enterobactin (FepA) (8). The crystal structure of FhuA demonstrates (9, 10) a porin-like protein, with a 22-stranded C-terminal β barrel domain that encloses a globular N-terminal cork domain. Ferrichrome binds to the receptor via extracellular loops and apical regions of the cork domain. The cork domain occludes the lumen of the barrel, preventing passive diffusion of ferrichrome. Characteristic of TonB-dependent OM receptors, FhuA possesses an N-terminal consensus sequence termed the Ton box (8TITVTA13) that was not visible in the reported three-dimensional structures of FhuA.

We crystallized a purified complex of TonB (residues 33 to 239) and FhuA (residues 1 to 725) and collected data on a single TonB-FhuA crystal (11). The TonB-FhuA crystal structure consists of one protomer of TonB in complex with one protomer of FhuA (Fig. 1). The model was refined at a resolution of 3.3 Å to Rcryst = 28.4% and Rfree = 32.9% (table S1). The overall folds of the FhuA and TonB protomers are in agreement with previously reported structural data. The N-terminal globular cork domain (residues 19 to 160) of FhuA is enclosed by the C-terminal β-barrel domain (residues 161 to 725). The cork domain possesses a four-stranded β sheet between residues 47 and 154. The barrel domain is composed of 22 antiparallel β strands. The β strands terminate in 11 long loops on one face of the barrel and 10 short turns on the other face (Fig. 1A). Given the membrane topology of FhuA, the loops and turns are exposed to the extracellular environment and periplasm, respectively. A molecule of ferricrocin was observed bound to FhuA in a position not much different from that observed for the ligand-loaded FhuA structure 1QFF.

Fig. 1.

Overall structure of the TonB-FhuA complex. (A) Cartoon representation of TonB residues 158 to 235 complexed to FhuA. β strands are indicated as flat arrows; helices are indicated as flat coils. View is along a plane parallel to the OM. Horizontal bars delineate approximate OM boundaries. Arrow indicates direction toward periplasm. TonB is bound at the periplasmic face of FhuA. The FhuA cork domain (residues 19 to 160) is colored green; remaining residues (8 to 18; 161 to 725) are colored blue. TonB residues are colored yellow. (B) View of the TonB-FhuA complex along the longitudinal axis of the FhuA barrel, looking down on the periplasm-exposed surface of the complex. TonB secondary-structure elements (α1, α2, β1, β2, β3) are labeled. FhuA periplasmic turns 1, and 7 to 10 (T1, T7, T8, T9, T10), are also labeled for reference. (C) Electron density (blue) from a simulated-annealing composite omit 2FobsFcalc electron density map contoured at 1σ showing the extension of electron density from FhuA Ile9 to Gln18. FhuA residues between 8 and 18 are shown as sticks and colored by atom (carbon, white; nitrogen, blue; oxygen, red). FhuA cork domain residues (19 to 160) are shown as a green coil. FhuA barrel domain residues (161 to 725) are shown as a blue coil. TonB is shown as a yellow coil. TonB helices α1 and α2 are labeled for reference.

TonB residues 158 to 235 were observed in the TonB-FhuA structure (see supporting online text). The fold of the TonB protomer consists of a three-stranded β sheet (β1-β2 between residues 174 and 197, β3 between residues 223 and 231), a short helix α1 (residues 165 to 170), and a longer helix α2 (residues 203 to 210) (Fig. 1B). TonB interacts with the periplasm-exposed face of FhuA such that TonB helices α1 and α2 are oriented toward FhuA, with the TonB β sheet (composed of β1 to β3) distal to the FhuA barrel (Fig. 1A). The TonB protomer occupies approximately one-half of the periplasm-exposed surface area of FhuA and occludes the barrel lumen from the edge containing periplasmic turns 8 to 10 to a boundary delineated by a straight line between periplasmic turns 1 and 7 (Fig. 1B). Inspection of the TonB-FhuA structure relative to FhuA 1QFF revealed minor structural rearrangements [root mean square deviation (RMSD) = 0.23 Å] upon TonB binding. Structural changes proximal to the bound TonB are localized to the periplasmic turns of the FhuA barrel as well as the periplasm-exposed loop at the C terminus of the cork domain (fig. S1). Larger structural changes were observed in TonB upon complexation with FhuA. The overall Cα-Cα RMSD between FhuA-complexed TonB and TonB 1U07 is 0.93 Å. The largest main-chain deviations are localized mainly to the C terminus from residues Ile232 to Thr235. The Cα-Cα deviation relative to 1U07 at TonB Ile232 is 2.0 Å, whereas at Thr235 the Cα-Cα deviation is 3.5 Å.

We observed extensive and continuous electron density N-terminal to FhuA Glu19 and extending toward TonB. This unambiguous electron density allowed us to build FhuA residues 9 to 18 into the model, encompassing five of the six residues in the conserved FhuA Ton box consensus (Fig. 1C). The FhuA Ton box forms a parallel β interaction with β3 of the TonB C-terminal domain, extending the β sheet. FhuA residues Ile9, Thr10, Val11, and Ala13 interact with TonB residues Val225, Val226, Leu229, and Lys231, respectively (table S2). These interactions result in the formation of an interprotein β sheet with TonB β1 to β3 (Fig. 2A). The side chain of TonB Gln160, a residue previously shown to interact with the Ton box, was not visible in the electron density; a specific mode of interaction between this residue and the FhuA Ton box was not immediately apparent. However, sampling of the preferred side-chain rotamers at TonB residue 160 suggests a potential interaction with the FhuA Ton box via a hydrogen bond with the main-chain carbonyl oxygen atom of Thr12 (Fig. 2B). The residue immediately N-terminal to FhuA Thr12 is a position of conserved hydrophobicity. In FhuA this position is occupied by a valine residue. Multiple alignment of the Ton box regions of related TonB-dependent OM receptors (BtuB, FecA, FepA; fig. S2, red) reveals this position to be invariably occupied by valine. The hydrophobic environment at this position may contribute to the observed instability of TonB Gln160. The TonB-FhuA interprotein β sheet positions TonB α1 and α2, the two helices of the TonB C-terminal domain, such that they are oriented toward the periplasmic face of FhuA (Fig. 1C). The recently solved nuclear magnetic resonance structure of monomeric TonB (5) indicates that the C terminus of the protein folds back to form the fourth strand (β4) of the central β sheet of the C-terminal TonB domain. We do not observe TonB β4 in our structure. Instead, the C terminus of TonB extends away from the complex, such that the FhuA Ton box forms a parallel β interaction with TonB β3 (Fig. 2C). This observation clearly demonstrates that the complexation of TonB to FhuA occurs via strand exchange.

Fig. 2.

Interactions of the FhuA Ton box with the C-terminal domain of TonB. (A) The interprotein β sheet formed between the FhuA Ton box and the central β sheet of the TonB C-terminal domain. TonB and FhuA residues are shown as sticks. Coloring as in Fig. 1C. TonB strands β1 to β3 are labeled. FhuA periplasmic turns 8 and 9 (T8, T9) are also labeled for reference. (B) View along the plane of the interprotein β sheet showing potential hydrogen bonding between two rotamers of TonB Gln160 and the main-chain carbonyl oxygen atom of FhuA Thr12. FhuA residues are shown as sticks and are colored as in (A). TonB is shown as a yellow coil. Rotamers (“a” and “b”) of TonB Gln160 are shown as sticks colored by atom (carbon, green; nitrogen, blue; oxygen, red). TonB strands β1 to β3 are labeled. (C) Superposition of the TonB-FhuA crystal structure (TonB, yellow coil; FhuA, blue coil) and monomeric C-terminal TonB (PDB code 1XX3; pink coil). The Ton box and N terminus of FhuA, and the C terminus of TonB, from the crystal structure are labeled. FhuA periplasmic turns 7, 8, 9, and 10 (T7, T8, T9, T10) are also marked for reference. Yellow arrow indicates direction toward the C terminus of FhuA-complexed TonB. Pink arrow indicates direction toward the C terminus of monomeric TonB.

Our in vitro surface plasmon resonance studies (12, 13) indicated that TonB-FhuA complexation involves a kinetically limiting conformational rearrangement. The present structure showing exchange of TonB β4 with the FhuA Ton box corroborates these biophysical observations and suggests that formation of the interprotein β sheet is the initial committed step of TonB-FhuA complexation. The complex that we observe in this structure is likely the 1:1 high-affinity complex identified by biophysical methods. Interactions between a FhuA Ton box peptide and residues from TonB β3 and β4 were recently reported (5). Ton box residues may therefore form low-affinity encounter interactions with TonB β3 and β4 before formation of the stable interprotein β sheet. TonB Gln160 likely participates in these encounter interactions, perhaps facilitating displacement of TonB β4. A cysteine cross-linking study (14) mapped a complex network of interactions between TonB residues 159 and 164 such that each TonB residue interacted with multiple residues in the BtuB Ton box. This is consistent with TonB residues at or near Gln160 being involved in multiple transient encounter interactions before high-affinity complex formation.

The TonB-FhuA interface has a mean interfacial accessible surface area (ΔASA) (15) of 1299 Å2. The calculated shape correlation statistic (Sc) for this interface is 0.60, indicating that it has a surface complementarity similar to that observed for cork-barrel interfaces of TonB-dependent OM receptors (16). The TonB-FhuA interface is composed of a network of 17-residue pairs bound by hydrophilic interactions (table S2). All pairs were observed to have well-defined electron density for both FhuA and TonB side chains. Within this interface, a single electrostatic interaction occurs between FhuA cork residue Glu56 and TonB Arg166, located on TonB α1 (Fig. 3). A hydrogen bond is also formed between TonB Arg166 and the main-chain carbonyl oxygen atom of FhuA Ala26, located in the switch helix region. Ferrichrome binding to FhuA was previously observed to result in a 17.3 Å translocation of the FhuA N terminus and unwinding of the switch helix (9). Therefore, it appears that unwinding of the FhuA switch helix upon ligand binding occurs in order to stabilize FhuA interactions with TonB Arg166. FhuA barrel residues Ala591 and Asn594, located in or near periplasmic turn 8, also form hydrogen bonds with TonB Arg166.

Fig. 3.

Residues from the FhuA cork and barrel domains interacting with TonB Arg166. Cut-away view showing FhuA and TonB protomers in cartoon representation; β strands are shown as flat arrows, helices as flat coils. The FhuA cork domain (residues 19 to 160) is colored green; the remaining FhuA residues are colored blue. TonB is colored yellow. TonB Arg166 and interacting FhuA residues (Ala26, Glu56, Ala591, and Asn594) are shown as sticks colored by atoms (as in Fig. 1C). Strands of the central β sheet of the FhuA cork domain (β1 to β4) are labeled.

What does the crystal structure of the TonB-FhuA complex tell us about interactions of TonB with a cognate OM receptor and the transport of metal-chelated siderophore? Given our structural data, combined with findings from previous studies of TonB interactions with OM receptors in vitro and in vivo, we propose that the interprotein β sheet formed between the receptor Ton box and the TonB C-terminal domain is required to position TonB helix α1 proximal to the receptor cork domain. In the TonB-FhuA structure, this results in TonB Arg166 forming an electrostatic interaction with FhuA cork residue Glu56. Both TonB Arg166 (17) and FhuA Glu56 located in the TEE motif (16) are highly conserved residues, predicting functional importance of this TonB-FhuA ionic interaction. Molecular dynamics simulations of FhuA have suggested that cork domain solvation may lower the energy barrier for active transport of ferrichrome (18). It has recently been proposed that hydration of the central β sheet of the cork domain may render it prone to disruption by TonB through transmission of a relatively small force perpendicularly applied to the β strands of the cork domain (16). Given its position proximal to the central β sheet of the FhuA cork domain, TonB Arg166 is positioned to mediate a mechanical shearing or pulling force applied in trans to the central β sheet of the cork domain, resulting in its disruption. Localized unfolding of the cork domain would allow siderophore translocation into the periplasm, facilitating subsequent steps of the siderophore transport cycle.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 and S2

Tables S1 and S2


References and Notes

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