Outer Membrane Active Transport: Structure of the BtuB:TonB Complex

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Science  02 Jun 2006:
Vol. 312, Issue 5778, pp. 1396-1399
DOI: 10.1126/science.1127694


In Gram-negative bacteria, the import of essential micronutrients across the outer membrane requires a transporter, an electrochemical gradient of protons across the inner membrane, and an inner membrane protein complex (ExbB, ExbD, TonB) that couples the proton-motive force to the outer membrane transporter. The inner membrane protein TonB binds directly to a conserved region, called the Ton-box, of the transporter. We solved the structure of the cobalamin transporter BtuB in complex with the C-terminal domain of TonB. In contrast to its conformations in the absence of TonB, the Ton-box forms a β strand that is recruited to the existing β sheet of TonB, which is consistent with a mechanical pulling model of transport.

In addition to an inner (plasma) membrane, Gram-negative bacteria have an outer membrane that affords additional environmental protection to the organism. Porins, which are β barrel proteins that typically function as diffusion pores, permit passive transport across the outer membrane of molecules with molecular weights ∼600 daltons or less (1). However, bacteria, like other organisms, also require molecules that are larger and/or are present in the extracellular milieu at low concentration. Specifically, Gram-negative bacteria require iron, which is often taken up in the form of iron-siderophore complexes (2), as well as other organometallic compounds such as cobalamins (e.g., cyanocobalamin, vitamin B12) (3). Because a reduction of iron uptake correlates with a decrease in bacterial virulence (4), these transport systems are an attractive target for antibacterial drug discovery.

The uptake of scarce nutrients across the outer membrane is performed by a specialized active transport system that requires three components: specialized outer membrane transport proteins, an inner membrane multiprotein complex, and the inner membrane proton-motive force (pmf) to drive active transport (5). The outer membrane transporters have a common architecture of a 22-stranded β barrel situated in the membrane, long extracellular loops, short periplasmic turns, and a distinctive luminal domain (6). This luminal domain, composed of the N-terminal portion of the transporter, forms a globular-like domain that occludes the barrel. The inner membrane protein complex consists of the proteins ExbB, ExbD, and TonB. ExbB and ExbD are homologous to the MotA and MotB “stator” proteins of the bacterial flagellar motor (7). The protein TonB—which has a single putative transmembrane helix, a proline-rich linker region, and a periplasmic C-terminal domain—couples the inner membrane pmf to the outer membrane transporter. Multiple structures of the mixed α helical/β sheet C-terminal domain of TonB have been determined (8). TonB-dependent outer membrane transporters have a conserved motif, the Ton-box (9, 10), that interacts with TonB during the active transport cycle. Deletion (or certain mutations) of the Ton-box abrogate transport but do not affect substrate binding (3). The molecular mechanism of TonB-dependent outer membrane active transport is not known. Conformational change of the luminal domain to open a permeation path for substrate must occur, but whether the domain remains within the barrel or undergoes partial or full removal is not known. The presence of protein components in both bacterial membranes is suggestive of an “action-at-a-distance” mechanical pulling model, but compelling experimental evidence is lacking. In order to obtain additional information on the nature of TonB-dependent outer membrane active transport, we solved the structure of the C-terminal domain of TonB in complex with the cobalamin transporter BtuB.

BtuB and a C-terminal domain of TonB (residues 147 to 239) from Escherichia coli, were separately expressed and purified; the complex was made by combining BtuB and TonB in a molar ratio of ∼1:5 in the presence of the substrate cyanocobalamin (vitamin B12) and excess calcium. The structure was solved to 2.1 Å by molecular replacement using the structure of substrate-bound BtuB [Protein Data Bank (PDB) accession number 1NQH] (11) as the search model; the final crystallographic R and free-R factors are 0.192 and 0.251, respectively (12). The structure that we obtained is a 1:1 complex of BtuB:TonB (Fig. 1, A and B, and fig. S1). TonB is bound to the bottom (i.e., periplasmic-facing side) of BtuB, and covers approximately one-half of the bottom surface of BtuB (Fig. 1A). Compared with BtuB alone, the major structural changes of BtuB in the complex are in the conformations of its Ton-box (residues 6 to 12) and the residues (13 to 21) linking it with the luminal domain. The Ton-box in BtuB and other transporters has been observed to be ordered (but lacking regular secondary structure), or to be disordered (6). In the complex, the Tonbox of BtuB is recruited to form a parallel β strand with the three-stranded β sheet of TonB (Fig. 1B). A charged residue in TonB, Arg158, makes a salt bridge with Asp6 of the Ton-box. The Ton-box:TonB interaction is similar to that observed between the C-terminal domain of the bacterial periplasmic-spanning protein TolA and the bacteriophage g3p protein (fig. S3) (12, 13). The structure of TonB in the complex is very similar to those determined by x-ray crystallography (PDB accession number 1UO7) (14) and solution nuclear magnetic resonance (NMR) spectroscopy (PDB accession number 1XX3) (15), with root mean square deviation of common α carbons of 0.8 and 1.1 Å, respectively (fig. S2). The BtuB:TonB interface has an area of 1481 Å2; the Ton-box:TonB interface contributes 542 Å2 (36%). In addition to the Ton-box and its linker (and some extracellular loops shifted by crystal contacts), the largest structural changes are in one of the apical loops (residues 82 to 95) that bind the substrate (Fig. 1C) and in a portion of the luminal domain (residues 110 to 126) that interacts with the interior of the β barrel (Fig. 1D).

Fig. 1.

The BtuB:TonB complex. (A) Ribbon diagram of the BtuB:TonB complex with bound cyanocobalamin (vitamin B12) substrate shown as red spheres. In BtuB, the Ton-box (residues 6 to 12) is in blue; luminal domain (residues 13 to 137) in green; and β barrel (residues 138 to 594) in orange. The TonB C-terminal domain (residues 147 to 239, with residues 153 to 233 seen in the structure) is in pink. The view is from the periplasmic surface. (B) Ribbon diagram of the BtuB:TonB complex, cutaway to show the luminal domain and Ton-box:TonB interaction. The structure is oriented such that, with respect to the bacterium, up is extracellular, down is periplasm-facing, and the β barrel is embedded within the outer membrane. (C) Conformational change in an apical substrate binding loop (residues 86 to 95) of BtuB in the BtuB:TonB complex. This loop connects β strands A and B of the luminal domain and contacts the substrate. In the presence of substrate (cyanocobalamin, red ball-and-stick), this loop (yellow sticks) is shifted ∼6 Å with respect to the loop in the substrate-free (apo) structure (cyan sticks). In the BtuB:TonB complex, this loop (green sticks) is moved ∼2 Å closer to the substrate relative to the substrate-bound structure. (D) Conformational change, of the highly-conserved RP box and latch motifs (residues 110 to 126) (21) in the BtuB:TonB complex. This region is shifted by ∼3 Å relative to structures without bound TonB. BtuB:TonB, green sticks; substrate-bound BtuB, yellow sticks; and substrate-free BtuB, cyan sticks. All figures except fig. S4 made with PyMOL (33).

Cysteine-scanning crosslinking studies of interactions between TonB and BtuB (16), and between TonB and the ferric citrate transporter FecA (17), have identified pairwise interactions between these proteins. The structure of the complex is consistent with these interactions (Fig. 2A). In the TonB:BtuB crosslinking experiments, TonB-Gln160 interacts strongly with BtuB-Leu8 and -Val10; TonB-Gln162 with BtuB-Leu8 and -Ala12; and TonB-Tyr163 with BtuB-Ala12. Mapping the FecA Ton-box to BtuB indicates that crosslinking occurs most strongly at TonB-Gln160 with BtuB-Asp6, -Leu8, and -Val10, and TonB-Gln162 with BtuB-Asp6, -Val9, and -Val10. A solution NMR study (15) characterized the chemical shift changes in TonB when Ton-box peptides bound to it; the largest changes (in those residues that are present in our structure) are in TonB residues Gln160, Ala167, Gly174, Ile232, and Asn233. The structure of the complex is consistent with these interactions (Fig. 2B). A surface representation of the TonB:Ton-box region of the structure illustrates additional constraints on the Ton-box sequence (Fig. 2C). In the Ton-box:TonB parallel β sheet, formed by BtuB residues 6 to 12 and TonB residues 226 to 232, the even-numbered residues of the Ton-box participate in hydrogen bonding whereas the odd-numbered residues do not. Proline cannot form interstrand hydrogen bonds; therefore, its substitution at even positions would be deleterious to parallel β sheet formation, whereas odd-position substitution would be tolerated. This prediction rationalizes a series of proline-substitution loss-of-function mutations characterized in BtuB (18). L8P (where Leu8 is replaced by Pro) and V10P are nonfunctional; T7P, V9P, and T11P mutants have wild-type activity (19). The existence of TonB gain-of-function mutants Q160K and Q160L (20), which compensate for the nonfunctional BtuB L8P mutation, suggests that transport can occur with a destabilized Ton-box:TonB β sheet if compensated by other Ton-box:TonB interactions (12).

Fig. 2.

Ton-box:TonB interactions and properties. (A) Ton-box:TonB cysteine-scanning cross-linking data mapped onto the structure. The Ton-box of BtuB (Asp6, Thr7, Leu8, Val9, Val10, Thr11, and Ala12) is shown as blue sticks; TonB residues Arg158, Asn159, Gln160, Pro161, Gln162, Tyr163, and Pro164 are shown as green sticks. Dashed lines depict disulfide crosslinks observed between BtuB and TonB (16); solid lines depict disulfide crosslinks observed between FecA and TonB (17) (with the residues of the FecA Ton-box aligned with the BtuB Ton-box). The salt-bridge between TonB Arg158 and Ton-box Asp6 can be seen. (B) NMR chemical-shift data obtained from Ton-box peptide binding to TonB (15), mapped onto the structure. The Ton-box of BtuB (Asp6, Thr7, Leu8, Val9, Val10, Thr11, and Ala12) is shown in blue ribbon; TonB is shown in orange ribbon. The residues (Gln160, Ala167, Gly174, Ile232, and Asn233) present in our structure that displayed the five largest chemical shifts are shown as green side-chain sticks (except for Gly174, where the backbone is colored green). (C) Packing of the Ton-box with TonB. The Ton-box (yellow sticks with black mesh) packs within a crevice formed by a β strand and coil of TonB (surface representation).

The core of the luminal domain of TonB-dependent transporters is a four-stranded β sheet. In a previous publication (21), we noted the resemblance of this core domain to that used in single-molecule mechanical unfolding experiments. In particular, in these experiments there is a marked anisotropy in the amount of force required to unfold the β sheet; perpendicular to the β strands of the sheet requires negligible force and parallel requires much greater force (22, 23). The orientation of the luminal domain core suggested that only a very modest pulling force by TonB would suffice to affect its large conformational change or (partial or full) unfolding. The Ton-box:TonB complex is a four-stranded β sheet core; however, its orientation is rotated nearly 90° with respect to the luminal domain core (Fig. 3). Thus, a (much) larger force might be necessary to disrupt its interstrand interaction. We speculate that if TonB functions by application of a pulling force during the transport cycle, then conformational change of the luminal domain would occur before disengagement of TonB from the transporter. Clearly, such a hypothesis requires verification by multiple experimental approaches.

Fig. 3.

Dissimilar orientations of the β sheet cores of the luminal domain of BtuB and of the Ton-box:TonB domain. The luminal domain is shown in green; TonB is shown in pink; the Ton-box of BtuB is shown in blue; the bound cyanocobalamin substrate is shown as red spheres. The black arrow, pointing into the periplasmic space and toward the plasma membrane in the bacterial cell, indicates the approximate direction in which intact TonB might exert a pulling force during the transport cycle. The luminal domain β sheet is oriented such that a small force applied in the indicated direction, roughly perpendicular to the β strands, might suffice to affect substantial conformational change.

Binding of TonB to the outer membrane transporter leads to a mechanical pulling force or some other type of interaction that drives active transport. As the N-terminal luminal domain of the transporter occludes the channel, conformational rearrangement must occur to allow vectorial substrate transport through the lumen of the β barrel. The luminal domain, through alteration of its size and shape, may remain within the β barrel and expose a permeation pathway; alternatively, the luminal domain may exit the β barrel (possibly by an unfolding process) (24). Two studies on disulfide linking of luminal and β barrel domains in the hydroxamate transporter FhuA (25, 26) are consistent with a “partial unfolding model” of transport. In Endriß et al. (25), disulfide formation between residue 27 (at the N terminus) and residue 533 of the β barrel yields a mutant lacking transport activity. Conversely, Eisenhauer et al. (26) observe that mutants containing disulfides between residues 109 and 356, or between residues 112 and 383, still transport substrate. Approximately 80 residues of the luminal domain (N terminus, two β strands, and a long linker domain between them) separate the TonB:transporter interaction from where the luminal domain is tethered to the barrel. We interpret these results to indicate that affixing the luminal domain at (or near) its N terminus to the β barrel prevents any conformational change via TonB interaction. However, partial unfolding (or some other conformational change) in a portion of the luminal domain through TonB interaction is sufficient for substrate transport. The degree of unfolding required may be a function of the size of the substrate, which ranges from several hundred to more than a thousand daltons.

The structure of the BtuB:TonB complex provides a structural rationalization for the conservation of the Ton-box in TonB-dependent outer membrane transporters. Given that TonB binds to approximately one-half of the periplasmic surface of BtuB, our structure does not sterically exclude the possibility of two TonB molecules binding to the transporter during the transport cycle. TonB dimerization has been observed in vivo (27, 28), and a 2:1 complex of TonB and the ferricrocin transporter FhuA has been characterized in vitro (29). However, a second coincident TonB could not use the Ton-box in binding, so its affinity would be much lower (as has been observed). Binding of the substrate to the transporter likely induces an order-to-disorder transition in the Ton-box (8), and this disordered Ton-box is the recognition element for TonB. Because the TonB-transporter interaction is transient (i.e., TonB may interact serially with multiple outer membrane transporters to perform multiple transport cycles), the presence of disorder (high entropy) of the Ton-box is a useful strategy for obtaining high specificity without high affinity (30). The disordered Ton-box may be useful for sweeping out a larger volume within the periplasm, thus increasing the probability of encountering TonB. The conserved salt bridge between the Ton-box and TonB may be critical for longer range electrostatic attraction and nucleation of β strand formation of the Ton-box. Lastly, the structure of this complex, particularly the Ton-box:TonB region, may be useful in structure-based lead compound discovery for novel antibacterials. The addition of Ton-box peptides to media inhibits bacterial growth (31), and the expression of periplasmic domains of TonB inhibits transporter function (32). Both of these results indicate that disruption of the physiological TonB:Ton-box interface may provide a drug-discovery paradigm.

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