The Escherichia coli outer membrane cobalamin transporter BtuB: structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation

Gram-negative bacteria possess specialized active transport systems that function to transport organometallic cofactors or carriers, such as cobalamins, siderophores, and porphyrins, across their outer membranes. The primary components of each transport system are an outer membrane transporter and the energy-coupling protein TonB. In Escherichiacoli, the TonB-dependent outer membrane transporter BtuB carries out active transport of cobalamin (Cbl) substrates across its outer membrane. Cobalamins bind to BtuB with nanomolar affinity. Previous studies implicated calcium in high-affinity binding of cyanocobalamin (CN-Cbl) to BtuB. We previously solved four structures of BtuB or BtuB complexes: an apo-structure of a methionine-substitution mutant (used to obtain experimental phases by selenomethionine single-wavelength anomalous diffraction studies); an apo-structure of wild-type BtuB; a binary complex of calcium and wild-type BtuB; and a ternary complex of calcium, CN-Cbl and wild-type BtuB. We present an analysis of the binding of calcium in the binary and ternary complexes, and show that calcium coordination changes upon substrate binding. High-affinity CN-Cbl binding and calcium coordination are coupled. We also analyze the binding mode of CN-Cbl to BtuB, and compare and contrast this binding to that observed in other proteins that bind Cbl. BtuB binds CN-Cbl in a manner very different from Cbl-utilizing enzymes and the periplasmic Cbl binding protein BtuF. Homology searches of bacterial genomes, structural annotation based on the presence of conserved Cbl-binding residues identified by analysis of our BtuB structure, and detection of homologs of the periplasmic Cbl-binding binding protein BtuF enable identification of putative BtuB orthologs in enteric and non-enteric bacterial species.     

Identification of the Periplasmic Cobalamin-Binding Protein BtuF of Escherichia coli

Cells of Escherichia coli take up vitamin B(12) (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in SALMONELLA: The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His(6) tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.     

The β-barrel domain of FhuAΔ5-160 is sufficient for TonB-dependent FhuA activities of Escherichia coli

FhuA in the outer membrane of Escherichia coli serves as a transporter for ferrichrome, the antibiotics albomycin and rifamycin CGP4832, colicin M, and as receptor for phages T1, T5 and phi80. The previously determined crystal structure reveals that residues 160-714 of the mature protein form a beta-barrel that is closed from the periplasmic side by the globular N-proximal fragment, residues 1-159, designated the cork. In this study, deletion of the cork resulted in a stable protein, FhuADelta5-160, that was incorporated in the outer membrane. Cells that synthesized FhuADelta5-160 displayed a higher sensitivity to large antibiotics such as erythromycin, rifamycin, bacitracin and vancomycin, and grew on maltotetraose and maltopentaose in the absence of LamB. Higher concentrations of ferrichrome supported growth of a tonB mutant that synthesized FhuADelta5-160. These results demonstrate non-specific diffusion of compounds across the outer membrane of cells that synthesize FhuADelta5-160. However, growth of a FhuADelta5-160 tonB wild-type strain occurred at low ferrichrome concentrations, and ferrichrome was transported at about 45% of the FhuA wild-type rate despite the lack of ferrichrome binding sites provided by the cork. FhuADelta5-160 conferred sensitivity to the phages and colicin M at levels similar to that of wild-type FhuA, and to albomycin and rifamycin CGP 4832. The activity of FhuADelta5-160 depended on TonB, although the mutant lacks the TonB box (residues 7-11) previously implicated in the interaction of FhuA with TonB. CCCP inhibited tonB-dependent transport of ferrichrome through FhuADelta5-160. FhuADelta5-160 still functions as a specific transporter, and sites in addition to the TonB box are involved in the TonB-mediated response of FhuA to the proton gradient of the cytoplasmic membrane. It is proposed that TonB interacts with the TonB box of FhuA and with the beta-barrel to release ferrichrome from the FhuA binding sites and to open the channel in FhuA. For transport of ferrichrome through the open channel of FhuADelta5-160, interaction of TonB with the beta-barrel is sufficient to release ferrichrome from the residual binding sites at the beta-barrel and to induce the active conformation of the L4 loop at the cell surface for infection by the TonB-dependent phages T1 and phi80.     

TonB-dependent receptors—structural perspectives

Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel β-barrel traverses the outer membrane and adjacent β-strands are connected by extracellular loops and periplasmic turns. Located inside the β-barrel is the plug domain, composed primarily of a mixed four-stranded β-sheet and a series of interspersed α-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.     

TonB-dependent receptors-structural perspectives

Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel beta-barrel traverses the outer membrane and adjacent beta-strands are connected by extracellular loops and periplasmic turns. Located inside the beta-barrel is the plug domain, composed primarily of a mixed four-stranded beta-sheet and a series of interspersed alpha-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.     

FhuA barrel-cork hybrids are active transporters and receptors

The crystal structure of Escherichia coli FhuA reveals a beta-barrel domain that is closed by a globular cork domain. It has been assumed that the proton motive force of the cytoplasmic membrane through the interaction of the TonB protein with the TonB box of the cork opens the FhuA channel. Yet, deletion of the cork results in an FhuA derivative, FhuADelta5-160, that still displays TonB-dependent substrate transport and phage receptor activity. To investigate this unexpected finding further, we constructed FhuADelta5-160 derivatives of FhuA proteins from Salmonella paratyphi B, Salmonella enterica serovar Typhimurium, and Pantoea agglomerans. The FhuADelta5-160 proteins inserted correctly into the outer membrane, and with the exception of the P. agglomerans protein, transported ferrichrome and albomycin. FhuA hybrids consisting of the beta-barrel of one strain and the cork of another strain were active and showed higher TonB-dependent ferrichrome transport rates than the corkless derivatives. Exceptions were the E. coli beta-barrel/Salmonella serovar Typhimurium cork hybrid protein and the Salmonella serovar Typhimurium beta-barrel/P. agglomerans cork hybrid protein, both of which were less active than the beta-barrels alone. Each of the FhuA mutant proteins displayed activity for each of their ligands, except for phage T5, only when coupled to TonB. The hybrid FhuA proteins displayed a similar activity with the E. coli TonB protein as with their cognate TonB proteins. Sensitivity to phages T1, T5, and phi80, rifamycin CGP 4832, and colicin M was determined by the beta-barrel, whereas sensitivity to phage ES18 and microcin J25 required both the beta-barrel and cork domains. These results demonstrate that the beta-barrel domain of FhuA confers activity and specificity and responds to TonB and that the cork domains of various FhuA proteins can be interchanged and contribute to the activities of the FhuA hybrids.     

Sequence changes in the ton box region of BtuB affect its transport activities and interaction with TonB protein

Uptake of cobalamins by the transporter protein BtuB in the outer membrane of Escherichia coli requires the proton motive force and the transperiplasmic protein TonB. The Ton box sequence near the amino terminus of BtuB is conserved among all TonB-dependent transporters and is the only known site of mutations that confer a transport-defective phenotype which can be suppressed by certain substitutions at residue 160 in TonB. The crystallographic structures of the TonB-dependent transporter FhuA revealed that the region near the Ton box, which itself was not resolved, is exposed to the periplasmic space and undergoes an extensive shift in position upon binding of substrate. Site-directed disulfide bonding in intact cells has been used to show that the Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented and specific manner to form BtuB-TonB heterodimers that are stimulated by the presence of transport substrate. Here, replacement of Ton box residues with proline or cysteine revealed that residue side chain recognition is not important for function, although replacement with proline at four of the seven Ton box positions impaired cobalamin transport. The defect in cobalamin utilization resulting from the L8P substitution was suppressed by cysteine substitutions in adjacent residues in BtuB or in TonB. This suppression did not restore active transport of cobalamins but may allow each transporter to function at most once. The uncoupled proline substitutions in BtuB markedly affected the pattern of disulfide bonding to TonB, both increasing the extent of cross-linking and shifting the pairs of residues that can be joined. Cross-linking of BtuB and TonB in the presence of the BtuB V10P substitution became independent of the presence of substrate, indicating an additional distortion of the exposure of the Ton box in the periplasmic space. TonB action thus requires a specific orientation for functional contact with the Ton box, and changes in the conformation of this region block transport by preventing substrate release and repeated transport cycles.     

Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.

The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.     

TonB or not TonB: is that the question?

Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.     

Enhanced binding of TonB to a ligand-loaded outer membrane receptor: role of the oligomeric state of TonB in formation of a functional FhuA.TonB complex

The ferric hydroxymate uptake (FhuA) receptor from Escherichia coli facilitates transport of siderophores ferricrocin and ferrichrome and siderophore-antibiotic conjugates such as albomycin and rifamycin CGP 4832. FhuA is also the receptor for phages T5, T1, Phi80, UC-1, for colicin M and for the antimicrobial peptide microcin MccJ21. Energy for transport is provided by the cytoplasmic membrane complex TonB.ExbB.ExbD, which uses the proton motive force of the cytoplasmic membrane to transduce energy to the outer membrane. To accomplish energy transfer, TonB contacts outer membrane receptors. However, the stoichiometry of TonB. receptor complexes and their sites of interaction remain uncertain. In this study, analyses of FhuA interactions with two recombinant TonB proteins by analytical ultracentrifugation revealed that TonB forms a 2:1 complex with FhuA. The presence of the FhuA-specific ligand ferricrocin enhanced the amounts of complex but is not essential for its formation. Surface plasmon resonance experiments demonstrated that FhuA.TonB interactions are multiple and have apparent affinities in the nanomolar range. TonB also possesses two distinct binding regions: one in the C terminus of the protein, for which binding to FhuA is ferricrocin-independent, and a higher affinity region outside the C terminus, for which ferricrocin enhances interactions with FhuA. Together these experiments establish that FhuA.TonB interactions are more intricate than originally predicted, that the TonB.FhuA stoichiometry is 2:1, and that ferricrocin modulates binding of FhuA to TonB at regions outside the C-terminal domain of TonB.     

Transport of iron across the outer membrane

Summary The TonB protein is involved in energy-coupled receptor-dependent transport processes across the outer membrane. The TonB protein is anchored in the cytoplasmic membrane but exposed to the periplasmic space. To fulfill its function, it has to couple the energy-providing metabolism in the cytoplasmic membrane with regulation of outer membrane receptor activity. Ferrichrome and albomycin transport, uptake of colicin M, and infection by the phages T1 and80 occur via the same receptor, the FhuA protein in the outer membrane. Therefore, this receptor is particularly suitable for the study of energy-coupled TonB-dependent transport across the outer membrane. Ferrichrome, albomycin and colicin M bind to the FhuA receptor but are not released into the periplasmic space of unenergized cells, ortonB mutants. In vivo interaction between FhuA and TonB is suggested by the restoration of activity of inactive FhuA proteins, bearing amino acid replacements in the TonB box, by TonB derivatives with single amino acid substitutions. Point mutations in thefhuA gene are suppressed by point mutations in thetonB gene. In addition, naturally occurring degradation of the TonB protein and its derivatives is preferentially prevented in vivo by FhuA and FhuA derivatives where functional interaction takes place. It is proposed that in the energized state, TonB induces a conformation in FhuA which leads to the release of the FhuA-bound compounds into the periplasmic space. Activation of FhuA by TonB depends on the ExbBD proteins in the cytoplasmic membrane. They can be partially replaced by the TolQR proteins which show strong sequence similarity to the ExbBD proteins. A physical interaction of these proteins with the TonB protein is suggested by TonB stabilization through ExbB and TolQR. We propose a permanent or reversible complex in the cytoplasmic membrane composed of the TonB protein and the ExbBD/TolQR proteins through which TonB is energized.     

Dimerization of TonB is not essential for its binding to the outer membrane siderophore receptor FhuA of Escherichia coli

FhuA belongs to a family of specific siderophore transport systems located in the outer membrane of Escherichia coli. The energy required for the transport process is provided by the proton motive force of the cytoplasmic membrane and is transmitted to FhuA by the protein TonB. Although the structure of full-length TonB is not known, the structure of the last 77 residues of a fragment composed of the 86 C-terminal amino acids was recently solved and shows an intertwined dimer (Chang, C., Mooser, A., Pluckthun, A., and Wlodawer, A. (2001) J. Biol. Chem. 276, 27535-27540). We analyzed the ability of truncated C-terminal TonB fragments of different lengths (77, 86, 96, 106, 116, and 126 amino acid residues, respectively) to bind to the receptor FhuA. Only the shortest TonB fragment, TonB-77, could not effectively interact with FhuA. We have also observed that the fragments TonB-77 and TonB-86 form homodimers in solution, whereas the longer fragments remain monomeric. TonB fragments that bind to FhuA in vitro also inhibit ferrichrome uptake via FhuA in vivo and protect cells against attack by bacteriophage Phi80.     

Ligand-induced conformational change in the ferrichrome–iron receptor of Escherichia coli K-12

Ferrichrome–iron is actively transported across the outer membrane of Escherichia coli by the TonB-dependent receptor FhuA. To obtain FhuA in a form suitable for secondary-structure analyses, a hexahistidine tag was inserted into a surface-located site and the recombinant protein was purified by metal chelate chromatography. Functional studies indicated that the presence of the hexahistidine tag did not interfere with FhuA localization or with ligand-binding activity. Ferrichrome protected lysine 67 but not lysine 5 of purified recombinant FhuA from trypsinolysis. Results from trypsin digestion were interpreted as a conformational change in FhuA which had occurred upon ferrichrome binding, thereby preventing access of trypsin to lysine 67. Circular dichroism and Fourier transform infrared spectroscopy revealed a predominance of -sheet structure for the purified protein. In the presence of ferrichrome, FhuA exhibited a secondary structure and a thermostability which were similar to FhuA without ligand. The addition of ferrichrome to purified FhuA reduced the ability of certain anti-FhuA monoclonal antibodies to bind to the receptor. All antibodies which could in this manner discriminate between FhuA and FhuA bound to ferrichrome had their determinants within a loop which is toward the N-terminus and which is exposed to the periplasm. These data indicate that the binding of ferrichrome induces a structural change that is propogated across the outer membrane and results in an altered conformation of a periplasmically exposed loop of FhuA. It is proposed that by such an alteration of FhuA conformation, TonB is triggered to energize the active transport of the bound ligand across the outer membrane.     

Substrate-induced transmembrane signaling in the cobalamin transporter BtuB

The outer membranes of Gram-negative bacteria possess transport proteins essential for uptake of scarce nutrients. In TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, faces the periplasm and interacts with the inner membrane TonB protein to energize an active transport cycle. A critical mechanistic step is the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane signaling event increases the affinity of the transporter for TonB and enables active transport to proceed. We have solved crystal structures of BtuB, the outer membrane cobalamin transporter from Escherichia coli, in the absence and presence of cyanocobalamin (vitamin B(12)). In these structures, the Ton box is ordered and undergoes a conformational change in the presence of bound substrate. Calcium has been implicated as a necessary factor for the high-affinity binding (K(d) approximately 0.3 nM) of cyanocobalamin to BtuB. We observe two bound calcium ions that order three extracellular loops of BtuB, thus providing a direct (and unusual) structural role for calcium.     

Active transport of an antibiotic rifamycin derivative by the outer-membrane protein FhuA

BACKGROUND: FhuA, an integral membrane protein of Escherichia coli, actively transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane. The transport is coupled to the proton motive force, which energizes FhuA through the inner-membrane protein TonB. FhuA also transports the semisynthetic rifamycin derivative CGP 4832, although the chemical structure of this antibiotic differs markedly from that of ferric hydroxamates. RESULTS: X-ray crystallography revealed that rifamycin CGP 4832 occupies the same ligand binding site as ferrichrome and albomycin, thus demonstrating a surprising lack of selectivity. However, the binding of rifamycin CGP 4832 is deviant from the complexes of FhuA with hydroxamate-type ligands in that it does not result in the unwinding of the switch helix but only in its destabilization, as reflected by increased B factors. Unwinding of the switch helix is proposed to be required for efficient binding of TonB to FhuA and for coupling the proton motive force of the cytoplasmic membrane with energy-dependent ligand transport. The transport data from cells expressing mutant FhuA proteins indicated conserved structural and mechanistic requirements for the transport of both types of compounds. CONCLUSIONS: We conclude that the binding of rifamycin CGP 4832 destabilizes the switch helix and promotes the formation of a transport-competent FhuA-TonB complex, albeit with lower efficiency than ferrichrome. Active transport of this rifamycin derivative explains the 200-fold increase in potency as compared to rifamycin, which is not a FhuA-specific ligand and permeates across the cell envelope by passive diffusion only.     

Quantification of known components of the Escherichia coli TonB energy transduction system: TonB, ExbB, ExbD and FepA

The TonB-dependent energy transduction system couples cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes across the outer membrane in Gram-negative bacteria. In Escherichia coli, the primary players known in this process to date are: FepA, the TonB-gated transporter for the siderophore enterochelin; TonB, the energy-transducing protein; and two cytoplasmic membrane proteins with less defined roles, ExbB and ExbD. In this study, we report the per cell numbers of TonB, ExbB, ExbD and FepA for cells grown under iron-replete and iron-limited conditions. Under iron-replete conditions, TonB and FepA were present at 335 +/- 78 and 504 +/- 165 copies per cell respectively. ExbB and ExbD, despite being encoded from the same operon, were not equimolar, being present at 2463 +/- 522 and 741 +/- 105 copies respectively. The ratio of these proteins was calculated at one TonB:two ExbD:seven ExbB under all four growth conditions tested. In contrast, the TonB:FepA ratio varied with iron status and according to the method used for iron limitation. Differences in the method of iron limitation also resulted in significant differences in cell size, skewing the per cell copy numbers for all proteins.     

Transport across the outer membrane of Escherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane

Summary Point mutations in the “TonB box” offhuA were suppressed by point mutations in thetonB gene, suggesting both a functional and physical interaction between the FhuA receptor protein in the outer membrane and the TonB protein in the cytoplasmic membrane ofEscherichia coli K12. Mutations influA were classified into four types according to the extent by which they impaired mutant cells in their growth on ferrichrome as sole iron source and in their sensitivity to the antibiotic albomycin and to colicin M. ThetonB mutation with a glutamine to leucine replacement at position 165 was less efficient in restoring the FhuA functions than the glutamine to lysine exchange at the same position. The better the coupling between FhuA and TonB the poorer was the inhibition of phage T1 binding to FhuA by ferrichrome. A working model is proposed in which the TonB protein assumes different conformations in response to the energized state of the cytoplasmic membrane and thereby allosterically regulates the activity of the FhuA receptor. This model implies an intermembrane coupling between two proteins in adjacent membranes.