Structural characterization of Escherichia coli BamE, a lipoprotein component of the β-barrel assembly machinery complex

In Escherichia coli, the BAM complex catalyzes the essential process of assembling outer membrane proteins (OMPs). This complex consists of five proteins: one membrane-bound protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their importance in OMP biogenesis, there is currently a lack of functional and structural information on the BAM complex lipoproteins. BamE is the smallest but most conserved lipoprotein in the complex. The structural and dynamic properties of monomeric BamE (residues 21-133) were determined by NMR spectroscopy. The protein folds as two α-helices packed against a three-stranded antiparallel β-sheet. The N-terminal (Ser21-Thr39) and C-terminal (Pro108-Asn113) residues, as well as a β-hairpin loop (Val76-Gln89), are highly flexible on the subnanosecond time scale. BamE expressed and purified from E. coli also exists in a kinetically trapped dimeric state that has dramatically different NMR spectra, and hence structural features, relative to its monomeric form. The functional significance of the BamE dimer remains to be established. Structural comparison to proteins with a similar architecture suggests that BamE may play a role in mediating the association of the BAM complex or with the BAM complex substrates.     

Crystal structure of β-barrel assembly machinery BamCD protein complex

The β-barrel assembly machinery (BAM) complex of Escherichia coli is a multiprotein machine that catalyzes the essential process of assembling outer membrane proteins. The BAM complex consists of five proteins: one membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Here, we report the first crystal structure of a Bam lipoprotein complex: the essential lipoprotein BamD in complex with the N-terminal half of BamC (BamC(UN) (Asp(28)-Ala(217)), a 73-residue-long unstructured region followed by the N-terminal domain). The BamCD complex is stabilized predominantly by various hydrogen bonds and salt bridges formed between BamD and the N-terminal unstructured region of BamC. Sequence and molecular surface analyses revealed that many of the conserved residues in both proteins are found at the BamC-BamD interface. A series of truncation mutagenesis and analytical gel filtration chromatography experiments confirmed that the unstructured region of BamC is essential for stabilizing the BamCD complex structure. The unstructured N terminus of BamC interacts with the proposed substrate-binding pocket of BamD, suggesting that this region of BamC may play a regulatory role in outer membrane protein biogenesis.     

The reconstituted Escherichia coli Bam complex catalyzes multiple rounds of β-barrel assembly

β-Barrel proteins are folded and inserted into the outer membranes of Escherichia coli by the Bam complex. The Bam complex has been purified and functionally reconstituted in vitro. We report conditions for reconstitution that increase the folding yield 10-fold and allow us to monitor the time course of folding directly. We use these conditions to analyze the effect of a mutation in the Bam complex and to demonstrate the ability of the reconstituted complex to catalyze more than one round of substrate assembly.     

Crystal structure of BamD: an essential component of the β-Barrel assembly machinery of gram-negative bacteria

Folding and insertion of integral β-barrel proteins in the outer membrane (OM) is an essential process for Gram-negative bacteria that requires the β-barrel assembly machinery (BAM). Efficient OM protein (OMP) folding and insertion appears to require a consensus C-terminal signal in OMPs characterized by terminal F or W residues. The BAM complex is embedded in the OM and, in Escherichia coli, consists of the β-barrel BamA and four lipoproteins BamBCDE. BamA and BamD are broadly distributed across all species of Gram-negative bacteria, whereas the other components are present in only a subset of species. BamA and BamD are also essential for viability, suggesting that these two proteins constitute the functional core of the bacterial BAM complex. Here, we present the crystal structure of BamD from the thermophilic bacteria Rhodothermus marinus refined to 2.15 Å resolution. The protein contains five tetratricopeptide repeats (TPRs) organized into two offset tandems, each capped by a terminal helix. The N-terminal domain contains three TPRs and displays remarkable structural similarity with proteins that recognize targeting signals in extended conformations. The C-terminal domain harbors the remaining two TPRs and previously described mutations that impair binding to other BAM components map to this domain. Therefore, the structure suggests a model where the C-terminal domain provides a scaffold for interaction with BAM components, while the N-terminal domain participates in interaction with the substrates, either recognizing the C-terminal consensus sequence or binding unfolded OMP intermediates.     

The bacterial outer membrane β-barrel assembly machinery

β-Barrel proteins found in the outer membrane of Gram-negative bacteria serve a variety of cellular functions. Proper folding and assembly of these proteins are essential for the viability of bacteria and can also play an important role in virulence. The β-barrel assembly machinery (BAM) complex, which is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, has been the focus of many recent studies. This review summarizes the significant progress that has been made toward understanding the structure and function of the bacterial BAM complex.     

The essential β-barrel assembly machinery complex components BamD and BamA are required for autotransporter biogenesis

Autotransporter biogenesis is dependent upon BamA, a central component of the β-barrel assembly machinery (BAM) complex. In this report, we detail the role of the other BAM components (BamB-E). We identify the importance of BamD in autotransporter biogenesis and show that BamB, BamC, and BamE are not required.     

BamE structure: the assembly of β-barrel proteins in the outer membranes of bacteria and mitochondria

A study recently published in EMBO reports solves the solution structure of E. coli BamE, thus providing the basis for a better understanding of the mechanism of β-barrel assembly in bacterial and mitochondrial outer membranes.EMBO Rep (2011) advance online publication. doi: 10.1038/embor.2010.202β-barrel membrane proteins are found exclusively in the outer membrane of Gram-negative bacteria and the outer membranes of eukaryotic organelles of prokaryotic origin, mitochondria and chloroplasts. In contrast to the inner membrane, the bacterial outer membrane is an asymmetrical bilayer that consists mainly of lipopolysaccharides in the outer leaflet and phospholipids in the inner leaflet. Bacterial β-barrel outer membrane proteins (OMPs) mediate many cellular functions, for example, passive or selective diffusion of small molecules through the β-barrel pores across the outer membrane. By contrast, only a few mitochondrial β-barrel outer membrane proteins (MBOMPs) have been identified so far. The central machineries that mediate insertion and assembly of OMPs/MBOMPs are the β-barrel assembly machine (BAM) complex in the bacterial outer membrane and the topogenesis of outer-membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex in the mitochondrial outer membrane (Knowles et al, 2009; Endo & Yamano, 2010; Stroud et al, 2010; Fig 1). However, the molecular mechanisms of β-barrel protein topogenesis in bacterial and mitochondrial outer membranes remain poorly understood.Open in a separate windowFigure 1β-barrel protein assembly in bacterial and mitochondrial outer membranes. (A) Bacteria. Ribbon models of the structures of the Sec complex, SurA, BamA (Clantin et al, 2007; Kim et al, 2007), BamE and OMP. The upper and lower inserts show the surface of BamE (residues 20–108; viewed after approximately 90° rotation of the ribbon model around the horizontal axis toward the reader). Residues important for BamD binding are shown in red and residues with NMR signals that were perturbed by BamD binding are shown in yellow. The residue (Phe 74) important for PG binding is shown in red and the residues with NMR signals that were perturbed by PG binding are shown in yellow. (B) Mitochondria. Ribbon models were drawn for the structures of small Tim and MBOMP. IM, inner membrane; IMS, intermembrane space; MBOMP, mitochondrial β-barrel outer membrane protein; OM, outer membrane; OMP, outer membrane protein; PG, phosphatidylglycerol; POTRA, polypeptide transport-associated domain.Bacterial OMPs are synthesized in the cytosol as precursor proteins with an amino-terminal signal sequence that guides the proteins to the Sec machinery for crossing the inner membrane and is cleaved off in the periplasm. Periplasmic chaperones then escort OMPs through the aqueous periplasmic space in a partly unfolded state. On reaching the outer membrane, OMPs assemble into a β-barrel structure and insert into the outer membrane with the help of the BAM complex. The bacterial OMP insertion pathway can be compared to the assembly pathway of MBOMPs from the mitochondrial intermembrane space into the outer membrane. MBOMPs are synthesized in the cytosol and imported into the intermembrane space by the outer membrane translocator TOM40. The subsequent chaperone-mediated escort across the intermembrane space and insertion into the outer membrane by the TOB complex is similar to the OMP assembly process. Notably, the BAM and TOB complexes share the homologous β-barrel proteins BamA and Tob55/Sam50, respectively, as the central components of their insertion machineries. The BAM complex in Escherichia coli consists of BamA (YaeT/Omp85) and four accessory lipoproteins: BamB (YfgL), BamC (NlpB), BamD (YfiO) and BamE (SmpA). BamA and BamD are essential for cell growth, yet deletion of dispensable BamB, BamC or BamE leads to outer membrane defects manifested in hypersensitivity to antibiotics. Although BamAB and BamCDE can form distinct subcomplexes, they become functional only after formation of the entire BAM complex with all five subunits (Hagan et al, 2010).In this issue of EMBO reports, Knowles et al (2011) solve the nuclear magnetic resonance (NMR) solution structure of E. coli BamE, which sheds light on the roles of one of the Bam subunits in β-barrel protein assembly. The structure of BamE consists of a three-stranded antiparallel β-sheet packed against a pair of α-helices (Fig 1).As the ΔbamE mutant cannot grow in the presence of vancomycin, the authors identify functionally important residues of BamE by testing the effects of amino-acid substitutions in BamE on its inability to complement the growth defects of ΔbamE, without destabilizing BamE itself. Many of the identified residues are conserved among BamE proteins from different organisms and map to a single surface area on BamE. Interestingly, NMR signals of the residues around this region are sensitive to the addition of micelles containing the lipid phosphatidylglycerol, but not phosphatidylethanolamine or cardiolipin. In parallel, the authors analyse perturbation of the NMR spectra of BamE after the addition of purified BamB, C and D proteins. Only BamD affects the NMR spectra of BamE, and the BamD interacting region of BamE is found to overlap partly with the residues involved in phosphatidylglycerol binding. As the addition of BamD and phosphatidylglycerol have different effects on the NMR spectra of BamE, the binding of BamD and phosphatidylglycerol to BamE seem to take place simultaneously. What is the biological relevance of the observed interactions of BamE with both BamD and phosphatidylglycerol? As phosphatidylglycerol was found to help the insertion of OMPs into lipid liposomes (Patel et al, 2009), BamE might recruit the BAM complex through BamD to phosphatidylglycerol-rich regions in the outer membrane, or might directly recruit phosphatidylglycerol to form assembly points for OMP insertion and folding.What are the roles of other subunits of the BAM complex in β-barrel protein assembly? The essential subunit of the E. coli BAM complex BamA consists of two domains: the N-terminal polypeptide transport-associated (POTRA) domain repeat in the periplasm and the carboxy-terminal β-barrel domain, embedded in the outer membrane. The number of POTRA domains ranges from one to five in BamA homologues from different organisms. Of these POTRA domains, the one nearest to the C-terminal that is most connected to the β-barrel domain is essential for cell viability and its deletion leads to disassembly of the BAM complex (Kim et al, 2007). Structural studies of the E. coli BamA POTRA domains suggest that each POTRA domain has a common fold, whereas conformational rigidity might differ between inter-domain linkers (Gatzeva-Topalova et al, 2010; Fig 1). As individual POTRA domains have some affinity for unfolded substrate proteins, the periplasmic tandem POTRA repeat probably provides several substrate binding sites that slide the substrate progressively towards the BamA β-barrel domain. The β-barrel domain of BamA probably functions as a scaffold to facilitate the formation of β-strands, possibly through β-augmentation and subsequent spontaneous membrane insertion of the β-barrel. Yet, it is not clear whether this cradle for β-strand formation is provided by the pore formed within the monomer or oligomeric forms of the BamA β-barrel domain. Alternatively, membrane insertion and folding of OMPs might take place at the interface between BamA and the outer membrane lipid bilayer.How much of the β-barrel assembly process is conserved during the evolution of mitochondria from Gram-negative bacteria? Although the central subunits BamA and Tob55 of the BAM and TOB complexes are conserved, other subunits of these complexes are unrelated to each other. The POTRA domains of BamA are essential for recognition and assembly of bacterial OMPs, whereas that of Tob55 is dispensable for MBOMP assembly in the mitochondrial outer membrane. Nevertheless, the mitochondrial TOB complex facilitates assembly of bacterial OMPs at low efficiency (Walther et al, 2009) and, in turn, the bacterial BAM complex can mediate assembly of mitochondrial porin. Therefore, the basic mechanism of β-barrel assembly in the outer membranes of bacteria and mitochondria seems to be conserved. High-resolution structures of each component of the BAM and TOB complexes—including that of BamE in this study—will thus provide the basis for a better understanding of the mechanism of β-barrel assembly in evolutionarily related bacterial and mitochondrial outer membranes.     

Substitutions in the BamA β-barrel domain overcome the conditional lethal phenotype of a ΔbamB ΔbamE strain of Escherichia coli

BamA interacts with the BamBCDE lipoproteins, and together they constitute the essential β-barrel assembly machine (BAM) of Escherichia coli. The simultaneous absence of BamB and BamE confers a conditional lethal phenotype and a severe β-barrel outer membrane protein (OMP) biogenesis defect. Without BamB and BamE, wild-type BamA levels are significantly reduced, and the folding of the BamA β-barrel, as assessed by the heat-modifiability assay, is drastically compromised. Single-amino-acid substitutions in the β-barrel domain of BamA improve both bacterial growth and OMP biogenesis in a bamB bamE mutant and restore BamA levels close to the BamB(+) BamE(+) level. The substitutions alter BamA β-barrel folding, and folding in the mutants becomes independent of BamB and BamE. Remarkably, BamA β-barrel alterations also improve OMP biogenesis in cells lacking the major periplasmic chaperone, SurA, which, together with BamB, is thought to facilitate the transfer of partially folded OMPs to the soluble POTRA (polypeptide-transport-associated) domain of BamA. Unlike the bamB bamE mutant background, the absence of BamB or SurA does not affect BamA β-barrel folding. Thus, substitutions in the outer membrane-embedded BamA β-barrel domain overcome OMP biogenesis defects that occur at the POTRA domain of BamA in the periplasm. Based on the structure of FhaC, the altered BamA residues are predicted to lie on a highly conserved loop that folds inside the β-barrel and in regions pointing outside the β-barrel, suggesting that they influence BamA function by both direct and indirect mechanisms.     

Surface-tethered planar membranes containing the β-barrel assembly machinery: a platform for investigating bacterial outer membrane protein folding

The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the β-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring, we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin, and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment.     

EscI: a crucial component of the type III secretion system forms the inner rod structure in enteropathogenic Escherichia coli

The T3SS (type III secretion system) is a multi-protein complex that plays a central role in the virulence of many gram-negative bacterial pathogens. This apparatus spans both bacterial membranes and transports virulence factors from the bacterial cytoplasm into eukaryotic host cells. The T3SS exports substrates in a hierarchical and temporal manner. The first secreted substrates are the rod/needle proteins which are incorporated into the T3SS apparatus and are required for the secretion of later substrates, the translocators and effectors. In the present study, we provide evidence that rOrf8/EscI, a poorly characterized locus of enterocyte effacement-encoded protein, functions as the inner rod protein of the T3SS of EPEC (enteropathogenic Escherichia coli). We demonstrate that EscI is essential for type III secretion and is also secreted as an early substrate of the T3SS. We found that EscI interacts with EscU, the integral membrane protein that is linked to substrate specificity switching, implicating EscI in the substrate-switching event. Furthermore, we showed that EscI self-associates and interacts with the outer membrane secretin EscC, further supporting its function as an inner rod protein. Overall, the results of the present study suggest that EscI is the YscI/PrgJ/MxiI homologue in the T3SS of attaching and effacing pathogens.     

TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein

Definitive identification of Treponema pallidum rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in T. pallidum with sequence homology to a Gram-negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modelling predicted that five polypeptide transport-associated (POTRA) domains comprise the N-terminus of TP0326, while the C-terminus forms an 18-stranded amphipathic β-barrel. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the β-barrel is responsible for the native protein's amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease-surface accessibility assay confirmed surface exposure. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in T. pallidum larger than that of Escherichia coli. Non-orthologous ancillary factors and self-association of TP0326 via its β-barrel may both contribute to the Bam complex. T. pallidum-infected rabbits mount a vigorous antibody response to both POTRA and β-barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochaete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity.     

Crystal Structure of BamB Bound to a Periplasmic Domain Fragment of BamA,the Central Component of the β-Barrel Assembly Machine

The β-barrel assembly machinery (BAM) mediates folding and insertion of β-barrel outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. BAM is a five-protein complex consisting of the β-barrel OMP BamA and lipoproteins BamB, -C, -D, and -E. High resolution structures of all the individual BAM subunits and a BamD-BamC complex have been determined. However, the overall complex architecture remains elusive. BamA is the central component of BAM and consists of a membrane-embedded β-barrel and a periplasmic domain with five polypeptide translocation-associated (POTRA) motifs thought to interact with the accessory lipoproteins. Here we report the crystal structure of a fusion between BamB and a POTRA3–5 fragment of BamA. Extended loops 13 and 17 protruding from one end of the BamB β-propeller contact the face of the POTRA3 β-sheet in BamA. The interface is stabilized by several hydrophobic contacts, a network of hydrogen bonds, and a cation-π interaction between BamA Tyr-255 and BamB Arg-195. Disruption of BamA-BamB binding by BamA Y255A and probing of the interface by disulfide bond cross-linking validate the physiological relevance of the observed interface. Furthermore, the structure is consistent with previously published mutagenesis studies. The periplasmic five-POTRA domain of BamA is flexible in solution due to hinge motions in the POTRA2–3 linker. Modeling BamB in complex with full-length BamA shows BamB binding at the POTRA2–3 hinge, suggesting a role in modulation of BamA flexibility and the conformational changes associated with OMP folding and insertion.     

Crystal structure of Escherichia coli manganese superoxide dismutase at 2.1-Å resolution

The three-dimensional structure of the manganese-dependent superoxide dismutase (MnSOD) from Escherichia coli has been determined by X-ray crystallography at 2.1?Å resolution. The protein crystallizes with two homodimers in the asymmetric unit, and a model comprising 6528 protein atoms (residues 1–205 of all four monomers), four manganese ions and 415 water molecules has been refined to an R factor of 0.188 (R _free 0.218). The structure shows a high degree of similarity with other MnSOD and FeSOD enzymes. The Mn centres are 5-coordinate, trigonal bipyramidal, with His26 and a solvent molecule, probably a hydroxide ion, as apical ligands, and His81, Asp167 and His171 as equatorial ligands. The coordinated solvent molecule is linked to a network of hydrogen bonds involving the non-coordinated carboxylate oxygen of Asp167 and a conserved glutamine residue, Gln146. The MnSOD dimer is notable for the way in which the two active sites are interconnected and a "bridge" comprising His171 of one monomer and Glu170 of the other offers a route for inter-site communication. Comparison of E. coli MnSOD and FeSOD (a) reveals some differences in the dimer interface, (b) yields no obvious explanation for their metal specificities, and (c) provides a structural basis for differences in DNA binding, where for MnSOD the groove formed by dimerization is complementary in charge and surface contour to B-DNA.     

Structure and function of BamE within the outer membrane and the β-barrel assembly machine

Insertion of folded proteins into the outer membrane of Gram-negative bacteria is mediated by the essential β-barrel assembly machine (Bam). Here, we report the native structure and mechanism of a core component of this complex, BamE, and show that it is exclusively monomeric in its native environment of the periplasm, but is able to adopt a distinct dimeric conformation in the cytoplasm. BamE is shown to bind specifically to phosphatidylglycerol, and comprehensive mutagenesis and interaction studies have mapped key determinants for complex binding, outer membrane integrity and cell viability, as well as revealing the role of BamE within the Bam complex.     

Crystal structure determination of the β-cyclodextrin-p-aminobenzoic acid inclusion complex from powder X-ray diffraction data

The 1:1 inclusion complex of β-cyclodextrin and p-aminobenzoic acid was prepared and characterized by TG-DTA. The crystal structure of the complex was solved directly from powder X-ray diffraction data using the direct space approach and refined using Rietveld refinement techniques. The complex crystallizes in monoclinic P2₁ space group, with unit cell parameters a = 20.7890 ?, b = 10.2084 ?, c = 15.1091 ?, β = 110.825°, V = 2997 ?³. The amino group is located at the wide side of the β-cyclodextrin cavity, forming hydrogen bonds with β-cyclodextrin, and the carboxyl group is located at the narrow side. The crystallographic data obtained from powder diffraction data were compared with the single crystallographic data, and the result shows that solving crystal structure of cyclodextrins inclusion complexes of such complexity is accessible to powder diffractionists to some extent.     

Crystal structure of the γ-cyclodextrin n-propanol inclusion complex; Correlation of α-, β-, γ-cyclodextrin geometries

Crystals of the hydrated n-propanol inclusion complex of γ-cyclodextrin (γ-CD; cyclo-octaamylose) have space group P4, a = b = 23.759(7), c = 23.069(7)Å and six quarter γ-CD per asymmetric unit. The structure was solved by YZARC and refined to R = 14% using 6300 X-ray counter data. The γ-CD are stacked, n-propanol (not located) occupies the channel-type cavity and 27 water sites populate interstices between stacks. Within the stacks γ-CD are arranged head-to-head as well as head-to-tail and H-bonded with O(2), O(3), O(6) hydroxyls. In the series α-,β-,γ-CD, angles C(1′)-O(4)-C(4) reduce from 119°-117.7°-112.6°, virtual O(4′)?O(4) distances increase 4.23-4.39-4.48 Å. intramolecular H-bonding distances O(2)?O(3) between adjacent glucoses, 3.00 Å in α-CD are wider than ～2.83 Å in β- and γ-CD, indicating a greater flexibility of the former.