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.     

Sequences of the Escherichia coli BtuB protein essential for its insertion and function in the outer membrane.

The Escherichia coli btuB gene encodes the outer membrane transporter for vitamin B12, the E colicins, colicin A, and bacteriophage BF23. Several series of mutant forms of BtuB resulting from the insertion of dipeptide sequences and from overlapping in-frame deletions and duplications were constructed. Strains expressing the variant genes in single and multiple copy numbers were analyzed for BtuB function, for the level of BtuB polypeptide in the outer membrane, and for changes in the outer membrane permeability barrier. Most dipeptide insertions had normal transport function and assembly in the membrane. Only 2 of the 27 deletions spanning residues 5 and 514 possessed transport function, and most of the remainder were not stably inserted in the membrane. Most duplications (19 of 21) retained transport function and were inserted in the outer membrane, although some were subject to proteolysis. Even long duplications containing as many as 340 repeated amino-terminal residues retained function, suggesting considerable plasticity in the sequence requirements for membrane insertion of BtuB. Expression of many deletion and duplication proteins conferred increased susceptibility to structurally unrelated inhibitors that are normally excluded by the outer membrane. These results could be consistent with the mutational disruption of extracellular loops or transmembrane segments of BtuB that constitute a gated channel, but the finding that alterations throughout the length of BtuB affect membrane permeability properties suggests that the altered proteins might perturb the outer membrane structure itself.     

Deletions or duplications in the BtuB protein affect its level in the outer membrane of Escherichia coli.

The Escherichia coli btuB product is an outer membrane protein that mediates the TonB-coupled active transport of cobalamins and the uptake of the E colicins and bacteriophage BF23. The roles of various segments of the BtuB protein in its function or cellular localization were investigated by analysis of several genetic constructs. Hybrid proteins in which various lengths from the amino terminus of BtuB were linked to alkaline phosphatase (btuB::phoA genes) were all secreted across the cytoplasmic membrane. The BtuB-PhoA proteins that carried up to 327 amino acids of BtuB appeared to reside in the periplasmic space, whereas hybrid proteins containing at least 399 amino acids of BtuB were associated with the outer membrane. Eleven in-frame internal deletion mutations that spanned more than half of the mature sequence were prepared by combining appropriate restriction fragments from btuB variants with 6-bp linker insertions. None of the deleted proteins was able to complement any BtuB functions, and only three of them were detectable in the outer membrane, suggesting that most of the deletions affected sequences needed for stable association with the outer membrane. Duplications covering the same portions of BtuB were prepared in the same manner. All of these partial duplication variants complemented all BtuB functions, although some gave substantially reduced levels of activity. These proteins were found in the outer membrane, although some were subject to proteolytic cleavage within or near the duplicated segment. These results indicate that the insertion of BtuB into the outer membrane requires the presence of several regions of teh BtuB protein and that the presence of extra or redundant segments of the protein can be tolerated during its insertion and function.     

Multiple extracellular loops contribute to substrate binding and transport by the Escherichia coli cobalamin transporter BtuB

The Escherichia coli outer membrane TonB-dependent transporters for iron complexes and cobalamins recognize their multiple and diverse substrates with high specificity and affinity. The X-ray crystallographic structures of several transporters show that the substrate-binding surfaces are comprised of residues from the internal globular domain and multiple extracellular loops. The extracellular loops on the N-terminal half of the transmembrane beta-barrel of the cobalamin transporter BtuB participate in binding of the cofactor calcium atoms and undergo substantial conformation changes upon substrate binding. The functional relevance of the five C-terminal loops was examined by examining the effects of short in-frame deletions. Each loop contributed in different ways to the binding of BtuB substrates. Deletions in loops 7, 8, 9, and 11 strongly decreased cobalamin binding and transport, whereas deletions in loops 8, 9, and 10 affected binding and entry of phage BF23. None of the loops were essential for the action of colicin E1 or E3, which is consistent with the crystallographic observation that the colicin E3 receptor-binding domain can contact almost all of the loops. A deletion in loop 9 or 11 eliminated the ability of cobalamin to inhibit the action of colicin E1. These phenotypes show that there are multiple independent binding elements and point out similarities and differences in binding properties among the TonB-dependent transporters.     

A requirement for calcium in the transport of cobalamin across the outer membrane of Escherichia coli

Cobalamin (Cbl) transport across the outer membrane of cells of Escherichia coli consists of high affinity Cbl binding to the btuB protein of the Cbl receptor, followed by the proton motive force- and tonB-dependent release of the Cbl into the periplasmic space. During a search for experimental conditions that would mimic this release in vitro with isolated cell envelope particles, we found that calcium was required for the high affinity Cbl binding, and subsaturating calcium concentrations resulted in the decreased affinity of the Cbl receptor for Cbl. The apparent affinity of the Cbl receptor for calcium (KD, approximately 30 nM at pH 6.6) decreased with decreasing pH, resulting in decreased affinity for Cbl at lower pH values. With suboptimal levels of calcium, Cbl binding was decreased by millimolar levels of magnesium.     

Structures of wild-type and mutant signal sequences of Escherichia coli ribose binding protein.

The structure of a chemically synthesized 25-residue-long functional signal peptide of Escherichia coli ribose binding protein was compared with that of a nonfunctional mutant-signal peptide using circular dichroism and two-dimensional 1H NMR in solvents mimicking the amphiphilic environments. The functional peptide forms an 18-residue-long alpha-helix starting from the NH2-terminal region and reaching to the hydrophobic stretch in a solvent consisting of 10% dimethylsulfoxide, 40% water, and 50% trifluoroethanol (v/v). The nonfunctional mutant peptide, which contains a Pro at position 9 instead of a Leu in the wild-type peptide, does not have any secondary structure in that solvent but forms a 12-residue-long alpha-helix within the hydrophobic stretch in water/trifluoroethanol (50:50, v/v) solvent. It seems that the Pro-9 residue in the nonfunctional peptide disturbs the helix propagation from the hydrophobic stretch to the NH2-terminal region. Because both of these peptides have stable helices within the hydrophobic stretch, it may be concluded that the additional 2 turns of the alpha-helix in the NH2-terminal region of the wild-type signal peptide is important for its function.     

Inhibition of secretion of a mutant lipoprotein across the cytoplasmic membrane by the wild-type lipoprotein of the Escherichia coli outer membrane.

A globomycin-resistant mutant of Escherichia coli was found to produce a precursor of the major outer membrane lipoprotein (prolipoprotein), in which the glycine residue at position 14 within the signal peptide was replaced by an aspartic acid residue. The same mutation has been reported by Lin et al. (Proc. Natl. Acad. Sci. U.S.A. 175:4891-4895, 1978). The structural gene of the mutant prolipoprotein was inserted into an inducible expression cloning vehicle. When the mutant prolipoprotein was produced in lipoprotein-minus host cells, 82% of the unprocessed protein was found in the membrane fraction, with the remaining 18% localized in the soluble fraction. However, when the production of the mutant prolipoprotein was induced in the wild-type lpp+ host cells, only 31% of the mutant prolipoprotein was found in the membrane fraction, leaving the remaining 69% in the soluble, cytoplasmic fraction. In addition, the assembly of the wild-type lipoprotein in these cells was not affected, whether the mutant prolipoprotein was produced or not. These results suggest that secretions of both mutant and wild-type prolipoproteins utilize the same component(s) responsible for the initial stages of secretion across the cytoplasmic membrane. However, it appears that the wild-type lipoprotein has a higher affinity for these components than does the mutant lipoprotein.     

Observations on the calcium dependence and reversibility of cobalamin transport across the outer membrane of Escherichia coli

The calcium dependence of cobalamin (Cbl) binding to the BtuB protein of Escherichia coli and the reversibility of its function in the transport of Cbl across the outer membrane have been examined. The results show that the two calcium-binding sites in BtuB that were identified previously by others are responsible for the calcium dependence of high affinity Cbl binding. The affinity of the pure BtuB protein for Cbl was approximately 1000-fold higher in the presence of saturating levels of calcium than in its absence. The affinities of BtuB for both Cbl and calcium were decreased by insertion of alanine residues at position 51 of the mature protein and were increased by several mutations and deletions in the TonB box. Experiments on the uptake of Cbl into the periplasmic space showed that this process is reversible and that the exit of Cbl back into the medium does not require the protonmotive force. Our interpretation of these results is that the role of the TonB-ExbB-ExbD complex, potentiated by the protonmotive force, is to reduce the affinity of the Cbl-binding site, thus increasing the rate of Cbl release into the periplasmic space. The evidence also indicates that access of the Cbl-binding site of BtuB to the periplasmic space does not require removal of the hatch domain from the barrel.     

The proton motive force drives the outer membrane transport of cobalamin in Escherichia coli.

Cells of Escherichia coli pump cobalamin (vitamin B12) across their outer membranes into the periplasmic space, and it was concluded previously that this process is potentiated by the proton motive force of the inner membrane. The novelty of such an energy coupling mechanism and its relevance to other outer membrane transport processes have required confirmation of this conclusion by studies with cells in which cobalamin transport is limited to the outer membrane. Accordingly, I have examined the effects of cyanide and of 2,4-dinitrophenol on cobalamin uptake in btuC and atp mutants, which lack inner membrane cobalamin transport and the membrane-bound ATP synthase, respectively. Dinitrophenol eliminated cobalamin transport in all strains, but cyanide inhibited this process only in atp and btuC atp mutant cells, providing conclusive evidence that cobalamin transport across the outer membrane requires specifically the proton motive force of the inner membrane. The coupling of metabolic energy to outer membrane cobalamin transport requires the TonB protein and is stimulated by the ExbB protein. I show here that the tolQ gene product can partly replace the function of the ExbB protein. Cells with mutations in both exbB and tolQ had no measurable cobalamin transport and thus had a phenotype that was essentially the same as TonB-. I conclude that the ExbB protein is a normal component of the energy coupling system for the transport of cobalamin across the outer membrane.     

Temperature-sensitive processing of outer membrane lipoprotein in an Escherichia coli mutant.

A mutant of Escherichia coli that accumulated prolipoprotein, a secretory precursor of the outer membrane lipoprotein, was isolated. The prolipoprotein accumulated in this mutant was modified by glyceride, but the in vitro cleavage of the signal peptide of the accumulated prolipoprotein was found to be temperature sensitive. The mutation appears to be located outside the gene for the lipoprotein, thus suggesting that the gene for the signal peptidase for the prolipoprotein was mutated.     

Strain and temperature-dependent variation in the amount of BtuB polypeptide in the Escherichia coli K-12 outer membrane

Abstract The outer membrane protein BtuB of Escherichia coli K-12 is the receptor for vitamin B₁₂; it is normally present in approx. 200 copies per cell. We describe here the conditions by which BtuB was readily observed in electropherograms of outer membrane preparations. These conditions are as follows. (1) Incorporation of 8 M urea in sodium dodecyl sulfate-polyacrylamide gel systems improved detection of the polypeptide. (2) In most E. coli K-12 strains examined, BtuB was most abundant when cells were grown at 27°C. This thermoregulation of BtuB was independent of envZ and envY , two regulatory genes for outer membrane proteins.     

Amino acid replacement in a mutant lipoprotein of the Escherichia coli outer membrane.

The primary structure of a mutant lipoprotein of the outer membrane of Escherichia coli was investigated. This mutant was previously described as a mutant that forms a dimer of the lipoprotein by an S-S bridge (H. Suzuki et al., J. Bacteriol. 127:1494-1501, 1976). The amino acid analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of tryptic peptides, it was found that the arginine residue at position 57 was replaced with a cysteine residue. The amino terminal structure of the mutant lipoprotein was found to be glycerylcysteine, as in the case of the wild-type lipoprotein. The present results show that the mutation that was previously determined to map at 36.5 min on the E. coli chromosome occurred in the structure gene (lpp) for the lipoprotein. This was further confirmed by the fact that a merodiploid carrying both lpp+ and lpp produces not only the wild-type lipoprotein but also the mutant lipoprotein.     

Role of a major outer membrane protein in Escherichia coli.

Mutants of Escherichia coli B/r lacking a major outer membrane protein, protein b, were obtained by selecting for resistance to copper. These mutants showed a decreased ability to utilize a variety of metabolites when the metabolites were present at low concentrations. Also, mutants of E. coli K-12 lacking proteins b and c from the outer membrane were shown to have an identical defect in the uptake of various metabolites. These results are discussed with regard to their implications as to the role of these proteins in permeability of the outer membrane,     

Topography of the insertion of LamB protein into the outer membrane of Escherichia coli wild-type and lac-lamB cells

The appearance of newly induced LamB protein at the cell surface of Escherichia coli was followed topographically by immuno-electron microscopy. LamB protein was induced in E. coli wild-type or lac-lamB cells for a short period of time (4 to 6 min), such that the overall level of LamB protein in induced cells was at least twofold higher than that in uninduced cells. Antibodies bound to LamB protein exposed at the cell surface were labeled with a protein A-gold probe, and the probe distribution in briefly induced cells was compared to that in uninduced cells. Analysis of large numbers of cells showed that newly inserted LamB protein appeared homogeneously over the entire cell surface, both in wild-type cells and in lac-lamB cells. A peak of insertion which was observed at the division site of the cell was also observed in the absence of induction and in control experiments in which a nonspecific probe was used. It is concluded therefore that insertion of LamB protein into the cell envelope of E. coli occurs at multiple sites over the entire cell surface. The average amount of LamB protein which appeared at the cell surface after induction was determined for various cell size classes. It was found that cells of various size classes all synthesized LamB protein after induction, indicating that synthesis of the protein was not restricted to cells in a particular stage of the cell cycle. However, the rate of LamB synthesis was found to vary during the cell cycle: this rate was constant regardless of cell size in nondividing cells, whereas it increased in dividing cells. It is concluded that the accumulation of newly induced LamB protein follows a linear pattern.     

Escherichia coli outer membrane protein K is a porin.

Protein K is an outer membrane protein found in pathogenic encapsulated strains of Escherichia coli. We present evidence here that protein K is structurally and functionally related to the E. coli K-12 porin proteins (OmpF, OmpC, and PhoE). Protein K was found to cross-react with antibody to OmpF protein and to share 8 out of 17 peptides in common with the OmpF protein. Strains that are OmpC porin- and OmpF porin- and contain protein K as their major outer membrane protein have increased rates of uptake of nutrients and a faster growth rate relative to the parental porin- strain. The protein K-containing strains are at least 1,000-fold more sensitive to colicins E2 and E3 than is the porin -deficient strain. These data suggest that protein K is a functional porin in E. coli. The porin function of protein K was also demonstrated in vitro, using black lipid membranes. Protein K increased the conductance in these membranes in discrete, uniform steps characteristic of channels with a size of about 2 nS.     

Point mutations in a conserved region (TonB box) of Escherichia coli outer membrane protein BtuB affect vitamin B12 transport.

Uptake of cobalamins and iron chelates in Escherichia coli K-12 is dependent on specific outer membrane transport proteins and the energy-coupling function provided by the TonB protein. The btuB product is the outer membrane receptor for cobalamins, bacteriophage BF23, and the E colicins. A short sequence near the amino terminus of mature BtuB, previously called the TonB box, is conserved in all tonB-dependent receptors and colicins and is the site of the btuB451 mutation (Leu-8----Pro), which prevents energy-coupled cobalamin uptake. This phenotype is partially suppressed by certain mutations in tonB. To examine the role of individual amino acids in the TonB box of BtuB, more than 30 amino acid substitutions in residues 6 to 13 were generated by doped oligonucleotide-directed mutagenesis. Many of the mutations affecting each amino acid did not impair transport activity, although some substitutions reduced cobalamin uptake and the Leu-8----Pro and Val-10----Gly alleles were completely inactive. To test whether the btuB451 mutation affects only cobalamin transport, a hybrid gene was constructed which encodes the signal sequence and first 39 residues of BtuB fused to the bulk of the ferrienterobactin receptor FepA (residues 26 to 723). This hybrid protein conferred all FepA functions but no BtuB functions. The presence of the btuB451 mutation in this fusion gene eliminated all of its tonB-coupled reactions, showing that the TonB box of FepA could be replaced by that from BtuB. These results suggest that the TonB-box region of BtuB is involved in active transport in a manner dependent not on the identity of specific side chains but on the local secondary structure.     

Eukaryotic precursor proteins are processed by Escherichia coli outer membrane protein OmpP.

A new specific endopeptidase that cleaves eukaryotic precursor proteins has been found in Escherichia coli K but not in E. coli B strains. After purification, protein sequencing and Western blotting, the endopeptidase was shown to be identical with E. coli outer membrane protein OmpP [Kaufmann, A., Stierhof, Y.-D. & Henning, U. (1994) J. Bacteriol. 176, 359-367]. Further characterization of enzymatic properties of the new peptidase was performed. Comparison of the cleavage specificities of the newly found endopeptidase and that of rat mitochondrial processing peptidase (MPP) showed that patterns of proteolytic cleavage on the investigated precursor proteins by both enzymes are similar. By using three mitochondrial precursor proteins, the specificity assigned to OmpP previously, a cleavage position between two basic amino-acid residues, was extended to a three amino-acid recognition sequence. Positions +1 to +3 of this extended recognition site consist of an amino-acid residue with a small aliphatic side chain such as alanine or serine, a large hydrophobic residue such as leucine or valine followed by an arginine residue. Additionally, structural motifs of the substrate seem to be required for OmpP cleavage.     

Mobility of BtuB and OmpF in the Escherichia coli outer membrane: implications for dynamic formation of a translocon complex

Diffusion of two Escherichia coli outer membrane proteins—the cobalamin (vitamin B12) receptor (BtuB) and the OmpF porin, which are implicated in the cellular import pathways of colicins and phages—was measured in vivo. The lateral mobility of these proteins is relevant to the mechanism of formation of the translocon for cellular import of colicins such as the rRNase colicin E3. The diffusion coefficient (D) of BtuB, the primary colicin receptor, complexed to fluorescent antibody or colicin, is 0.05 ± 0.01 μm²/s and 0.10 ± 0.02 μm²/s, respectively, over a timescale of 25-150 ms. Mutagenesis of the BtuB TonB box, which eliminates or significantly weakens the interaction between BtuB and the TonB energy-transducing protein that is anchored in the cytoplasmic membrane, resulted in a fivefold larger value of D, 0.27 ± 0.06 μm²/s for antibody-labeled BtuB, indicating a cytoskeletal-like interaction of TonB with BtuB. OmpF has a diffusion coefficient of 0.006 ± 0.002 μm²/s, ∼10-fold smaller than that of BtuB, and is restricted within a domain of diameter 100 nm, showing it to be relatively immobile compared to BtuB. Thus, formation of the outer membrane translocon for cellular import of the nuclease colicins is a demonstrably dynamic process, because it depends on lateral diffusion of BtuB and collisional interaction with relatively immobile OmpF.     

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.