Beta-barrel Assembly Machinery Research Articles

BAM folds OMPs using a budding mechanism.

In Gram-negative bacteria, the biogenesis of beta-barrel outer membrane proteins (OMPs) is mediated by the beta-barrel assembly machinery (BAM). The mechanism employed by BAM is complex and so far is not fully resolved. Here, we report the structures of BAM in nanodiscs, where we observe an outward-open state. Mutations in the barrel domain of BamA reveal that plasticity in BAM is essential, particularly along the lateral seam of the barrel domain, which is further supported by molecular dynamics simulations that show conformational dynamics in BAM are modulated by the accessory proteins. We also report a series of cryo-EM structures detailing the near complete folding of EspP by BAM, which reveals EspP threading from the underside of BAM and sequentially incorporating into the barrel domain of BamA. Together, our studies provide a complete view of OMP biogenesis by BAM, overwhelmingly supporting that OMPs are folded using the budding mechanism.

Thread-by-thread: modelling the intermediate steps of OMP insertion by BamA

Pathogenic Gram-negative bacteria utilize outer membrane proteins (OMPs) for a number of functions such as nutrient transport, signal transduction, and the export of virulence factors. These OMPs are folded and inserted into the outer membrane by the beta-barrel assembly machinery (BAM) complex, the core component of which is the OMP BamA. Previous computational, experimental, and structural studies have revealed that BamA possesses a dynamic lateral gate at the seam between its N- and C-terminal β-strands.

BamA is a pivotal protein in cell envelope synthesis and cell division in Deinococcus radiodurans

The beta-barrel assembly machinery (BAM) is an indispensable complex for protein transportation located at the outer membrane of bacteria. BAM is composed of five subunits (BamA-E) in the model bacterium Escherichia coli. DR_0379 is a BamA homolog in Deinococcus radiodurans, but the other subunits have not been detected in this species. In the present study, deletion of bamA resulted in decreased growth rate and altered morphology of D. radiodurans. ΔbamA cells underwent abnormal cell division, leading to aggregated bacteria of diverse size and shape, and the cell envelope was detached from the cell surface, resulting in reduced resistance to high ionic strength. Oxidative stress resistance was significantly enhanced in the mutant, which may be attributed to increased manganese ion concentration and Mn/Fe ratio. Numerous proteins were released into the medium from ΔbamA cells, including surface layer (S-layer) proteins and various transporters located in the periplasm and outer membrane. These results indicate that BamA affects the synthesis and assembly of the outer membrane and S-layer, and thereby influences material transport and cell division. The findings highlight the special functions of BamA in D. radiodurans, and promote our understanding of the multi-layer structure of the D. radiodurans cell envelope.

Mechanistic Insights into Outer Membrane Protein Folding

The beta-barrel outer membrane proteins (OMPs) of Gram-negative bacteria are essential for cell viability and survival, but the molecular mechanisms of their biogenesis remain poorly understood. OMPs are synthesised in the cytoplasm, translocated across the inner membrane and transported across the periplasm by the molecular chaperones Skp or SurA, before being inserted into the OM by the essential beta-barrel assembly machinery (BAM) complex (a 203 kDa heteropentameric membrane protein complex). The mechanisms by which Skp and SurA recognise OMPs and successfully deliver their cargos to BAM remain poorly understood. Questions about these mechanisms are especially interesting since the periplasm is devoid of ATP, and it is completely unknown how substrate binding and release are controlled and coordinated. Moreover, whilst several structures of the BAM complex have been solved recently, how OMPs are delivered to BAM from Skp/SurA and folded into the crowded outer membrane remain mysterious. We have been exploiting a range of biophysical methods to gain new insights into the mechanisms of OMP folding and assembly, and its assistance by chaperones and BAM. In this lecture I will describe our recent results using a variety of OMPs of different sizes combined with analysis using fluorescence kinetic assays, cryo-EM and non-covalent mass spectrometry. The results have revealed a new mechanism of Skp action and new insights into how BAM facilitates OMP folding into the bacterial outer membrane.

Effect of Lipid-Based Nanostructure on Protein Encapsulation within the Membrane Bilayer Mimetic Lipidic Cubic Phase Using Transmembrane and Lipo-proteins from the Beta-Barrel Assembly Machinery.

A fundamental understanding of the effect of amphiphilic protein encapsulation on the nanostructure of the bicontinuous cubic phase is crucial to progressing biomedical and biological applications of these hybrid protein-lipid materials, including as drug delivery vehicles, as biosensors, biofuel cells and for in meso crystallization. The relationship between the lipid nanomaterial and the encapsulated protein, however, remains poorly understood. In this study, we investigated the effect of incorporating the five transmembrane and lipo-proteins which make up the β-barrel assembly machinery from Gram-negative bacteria within a series of bicontinuous cubic phases. The transmembrane β-barrel BamA caused an increase in lattice parameter of the cubic phase upon encapsulation. By contrast, the mainly hydrophilic lipo-proteins BamB-E caused the cubic phase lattice parameters to decrease, despite their large size relative to the diameter of the cubic phase water channels. Analysis of the primary amino acid sequence was used to rationalize this effect, based on specific interactions between aromatic amino acids within the proteins and the polar-apolar interface. Other factors that were found to have an effect were lateral bilayer pressure and rigidity within the lipid bilayer, water channel diameter, and size and structure of the lipo-proteins. The data presented suggest that hydrophilic bioactive molecules can be selectively encapsulated within the cubic phase by using a lipid anchor or aromatic amino acids, for drug delivery or biosensing applications.

Evolution of the Translocation and Assembly Module (TAM).

Bacterial outer membrane proteins require the beta-barrel assembly machinery (BAM) for their correct folding and function. The central component of this machinery is BamA, an Omp85 protein that is essential and found in all Gram-negative bacteria. An additional feature of the BAM is the translocation and assembly module (TAM), comprised TamA (an Omp85 family protein) and TamB. We report that TamA and a closely related protein TamL are confined almost exclusively to Proteobacteria and Bacteroidetes/Chlorobi respectively, whereas TamB is widely distributed across the majority of Gram-negative bacterial lineages. A comprehensive phylogenetic and secondary structure analysis of the TamB protein family revealed that TamB was present very early in the evolution of bacteria. Several sequence characteristics were discovered to define the TamB protein family: A signal-anchor linkage to the inner membrane, beta-helical structure, conserved domain architecture and a C-terminal region that mimics outer membrane protein beta-strands. Taken together, the structural and phylogenetic analyses suggest that the TAM likely evolved from an original combination of BamA and TamB, with a later gene duplication event of BamA, giving rise to an additional Omp85 sequence that evolved to be TamA in Proteobacteria and TamL in Bacteroidetes/Chlorobi.

Structure and Function of the β-Barrel Assembly Machine and its Associated Chaperones

Membrane insertion of beta-barrel Outer Membrane Proteins (OMPs) is essential for Gram-Negative bacteria and requires the coordinated action of both periplasmic chaperones and the outer membrane complex known as beta-Barrel Assembly Machinery (BAM). BamA, a beta-barrel OMP itself, is the central component of BAM, which also has four associated lipoproteins: BamB, C, D and E. BamA and BamD are essential for viability and appear to be present in all bacteria with an outer membrane. In the current model, periplasmic chaperones prevent OMP aggregation in the periplasm and deliver their cargo to the BAM complex for insertion into the outer membrane. We have determined the crystal structure of the periplasmic chaperone Skp. With NMR and crosslinking experiments we show that Skp binds the transmembrane domain of OmpA, a model beta-barrel OMP, in its cavity maintaining it in an unfolded state while protecting it from aggregation. Interestingly, the periplasmic, non-membrane domain of OmpA is allowed to fold to its native conformation explaining the specific importance of Skp on the OMP biogenesis pathway. We also present high-resolution structures of the lipoproteins BamB, C and D as well as the periplasmic domain of BamA, which contains five POTRA motifs thought to organize the BAM complex and associate with nascent OMPs en route to insertion in the outer membrane. In addition, we report the first structure of a complex between BamB and a POTRA3-5 fragment of BamA that begins to illuminate the BAM complex architecture. Using complementary Small Angle X-ray Scattering and NMR we show that the BamA POTRA domain undergoes conformational transitions, which functional experiments show are crucial for its role in OMP folding and insertion.

The role of BamA in the biogenesis of beta-barrel membrane proteins

Beta-barrel membrane proteins are essential for nutrient import, signaling, motility, and survival. In Gram-negative bacteria, the beta-barrel assembly machinery (BAM) complex is responsible for the biogenesis of beta-barrel outer membrane proteins (OMPs), with homologous complexes found in mitochondria and chloroplasts. Despite their essential roles, exactly how these OMPs are formed remains unknown. The BAM complex consists of a central and essential component called BamA (an OMP itself) and four lipoproteins called BamB-E. While the structure of the lipoproteins have been reported, the structure of full length BamA has been elusive. Recently though, we described the structure of BamA from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane beta-barrel domain. Together, our crystal structures and molecule dynamics (MD) simulations revealed several structural features which gave clues to the mechanism by which BamA catalyzes beta-barrel assembly. The first is that the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the beta-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. Third, the beta-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane. And fourth, a surface exposed exit pore positioned above the lateral opening site which may play a role in the biogenesis of extracellular loops. In this presentation, the crystal structures and MD simulations of BamA will be presented along with our work looking at the role of these four structural features in the role of BamA within the BAM complex.

Structure and Function of the Beta-Barrel Assembly Machine and its Associated Chaperones

Membrane insertion of βeτα-barrel Outer Membrane Proteins (OMPs) is essential for Gram-Negative bacteria and requires the action of both periplasmic chaperones and the outer membrane complex known as beta-Barrel Assembly Machinery (BAM). Interfering with OMP transport and assembly may be an effective strategy for antibiotic development; much like β-lactams are effective by inhibiting cell wall synthesis. Therefore, it is crucial to understand the mechanisms by which periplasmic chaperones and the BAM complex work together to control OMP transport and assembly. The BAM complex is composed of BamA, a β-barrel OMP itself; and four associated lipoproteins: BamB, C, D and E. BamA and BamD are essential for viability and appear to be present in all bacteria with an outer membrane. Whereas the phylogenetic distribution of BamB, C and E is not well defined, these lipoproteins are also important for OMP biogenesis and their deletion in E. coli results in outer membrane permeability defects. In the current model, periplasmic chaperones prevent OMP aggregation in the periplasm and deliver their cargo to the BAM complex for insertion into the outer membrane. However, the molecular mechanisms mediating this process are still obscure. I present the crystal and NMR structures of BamB, BamD and BamC as well as the periplasmic domain of BamA. In addition we have just solved the structure of a complex between BamB and the periplasmic fragment of BamA, which begins to illuminate the architecture of this essential molecular machine. Using complementary biochemical and genetic experiments we show that the periplasmic domain of BamA is undergoes conformational transitions that are essential for its role in OMP folding and insertion. I'll present the implications of these structure/function studies for the mechanism of OMP folding and insertion.

Protein Plasticity and Protein-Lipid Interactions of the Beta-Barrel Assembly Machinery

Elucidating protein plasticity and protein-lipid interactions is critical for a detailed understanding of the workings of membrane protein machines. We study the 88 kDa membrane-embedded protein BamA, which is the main component of the beta-barrel assembly machinery (BAM) that folds and inserts outer membrane proteins in gram-negative bacteria. Using a combination of electron microscopy (EM), limited proteolysis and NMR we characterized the fold and dynamics of different BamA constructs and the role of the lipid bilayer for protein structure.Our NMR studies provide atomic insight into protein dynamics and plasticity of the periplasmic POTRA domains and their interactions with the transmembrane domain. Moreover, we studied the influence of lipid type and the lipid-to-protein ratio on the atomic (solid-state NMR) and nm (EM) scale. Taken together, our results provide first clues into the interplay between protein - protein and protein -lipid interactions that may be critical for BAM-mediated substrate insertion into the outer membrane.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Effects of the Periplasmic Domain of BamA, the Essential Transmembrane Protein of the Beta-Barrel Assembly Machinery (BAM), in Folding and Insertion of the Outer Membrane Protein A

In Escherichia coli, folding and insertion of outer membrane proteins (OMPs) is facilitated by the β-barrel assembly machinery (BAM), composed of the integral protein BamA and the four lipoproteins BamB, BamC, BamA and BamE. BamA consists of a transmembrane β-barrel and a soluble periplasmic domain (PD-BamA), comprised of five polypeptide transport-associated (POTRA) domains. Although it is known that PD-BamA is necessary for the activity of the BAM-complex, its mechanism is not understood. To examine whether PD-BamA promotes folding and insertion of OMPs into lipid membranes, we expressed and isolated it from E. coli. The secondary structure of PD-BamA was determined by CD spectroscopy to 36% α-helix, 15% β-strand, 20% β-turns, and 29% random coil. For OMP folding experiments, we isolated outer membrane protein A (OmpA) from E. coli in unfolded form in urea-solution. To investigate the effect of PD-BamA on the kinetics of OmpA folding into the model membranes different sets of experiments were performed. In parallel experiments, in the absence and presence of PD-BamA, the dependence of OmpA folding was examined by gel electrophoresis as described [1]. The effects of temperature, lipid composition, and lipid concentration on PD-BamA function were investigated. PD-BamA was found to promote folding of OmpA into lipid bilayers when phosphatidylglycerol (PG) was present in the membrane. No effect on the folding of OmpA could be observed in the absence of PG. Fluorescence spectroscopy showed that PD-BamA binds to lipid bilayers, but only when these contain PG. The kinetics of OmpA folding, determined at various PD-BamA/OmpA ratios, indicated a 1:1 stoichiometry of the interaction of PD-BamA and unfolded OmpA.[1] Kleinschmidt, J.H. (2003) Cell. Mol. Life Sci. 60, 1547-1558.

The beta-barrel outer membrane protein assembly complex of Neisseria meningitidis.

The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the beta-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components in Neisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As in E. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by its E. coli homolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. A bamC mutant showed only marginal OMP assembly defects, but the impossibility of creating a bamC bamE double mutant further indicated the function of BamC in OMP assembly. An mlp mutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex in N. meningitidis consists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly.

Membrane protein architects: the role of the BAM complex in outer membrane protein assembly

The folding of transmembrane proteins into the outer membrane presents formidable challenges to Gram-negative bacteria. These proteins must migrate from the cytoplasm, through the inner membrane and into the periplasm, before being recognized by the beta-barrel assembly machinery, which mediates efficient insertion of folded beta-barrels into the outer membrane. Recent discoveries of component structures and accessory interactions of this complex are yielding insights into how cells fold membrane proteins. Here, we discuss how these structures illuminate the mechanisms responsible for the biogenesis of outer membrane proteins.