Β-barrel Outer Membrane Proteins Research Articles

β-Barrel proteins tether the outer membrane in many Gram-negative bacteria.

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)1. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In E. coli, Braun’s lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability4 but most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present5. We use a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in stationary phase and tethering of the lipoprotein LimB similar throughout the cell-cycle. Using a genetic approach, we demonstrate that the cell-cycle dependent tethering of BbpA is partly dependent on a developmentally regulated L,D transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell-cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

Membrane thinning and lateral gating are consistent features of BamA across multiple species.

In Gram-negative bacteria, the folding and insertion of β-barrel outer membrane proteins (OMPs) to the outer membrane are mediated by the β-barrel assembly machinery (BAM) complex. Two leading models of this process have been put forth: the hybrid barrel model, which claims that a lateral gate in BamA’s β-barrel can serve as a template for incoming OMPs, and the passive model, which claims that a thinned membrane near the lateral gate of BamA accelerates spontaneous OMP insertion. To examine the key elements of these two models, we have carried out 45.5 μs of equilibrium molecular dynamics simulations of BamA with and without POTRA domains from Escherichia coli, Salmonella enterica, Haemophilus ducreyi and Neisseria gonorrhoeae, together with BamA’s homolog, TamA from E. coli, in their native, species-specific outer membranes. In these equilibrium simulations, we consistently observe membrane thinning near the lateral gate for all proteins. We also see occasional spontaneous lateral gate opening and sliding of the β-strands at the gate interface for N. gonorrhoeae, indicating that the gate is dynamic. An additional 14 μs of free-energy calculations shows that the energy necessary to open the lateral gate in BamA/TamA varies by species, but is always lower than the Omp85 homolog, FhaC. Our combined results suggest OMP insertion utilizes aspects of both the hybrid barrel and passive models.

Reversible autoinhibitory regulation of Escherichia coli metallopeptidase BepA for selective β-barrel protein degradation

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

Functions of the BamBCDE Lipoproteins Revealed by Bypass Mutations in BamA.

The heteropentomeric β-barrel assembly machine (BAM complex) is responsible for folding and inserting a diverse array of β-barrel outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria. The BAM complex contains two essential proteins, the β-barrel OMP BamA and a lipoprotein BamD, whereas the auxiliary lipoproteins BamBCE are individually nonessential. Here, we identify and characterize three bamA mutations, the E-to-K change at position 470 (bamAE470K ), the A-to-P change at position 496 (bamAA496P ), and the A-to-S change at position 499 (bamAA499S ), that suppress the otherwise lethal ΔbamD, ΔbamB ΔbamC ΔbamE, and ΔbamC ΔbamD ΔbamE mutations. The viability of cells lacking different combinations of BAM complex lipoproteins provides the opportunity to examine the role of the individual proteins in OMP assembly. Results show that, in wild-type cells, BamBCE share a redundant function; at least one of these lipoproteins must be present to allow BamD to coordinate productively with BamA. Besides BamA regulation, BamD shares an additional essential function that is redundant with a second function of BamB. Remarkably, bamAE470K suppresses both, allowing the construction of a BAM complex composed solely of BamAE470K that is able to assemble OMPs in the absence of BamBCDE. This work demonstrates that the BAM complex lipoproteins do not participate in the catalytic folding of OMP substrates but rather function to increase the efficiency of the assembly process by coordinating and regulating the assembly of diverse OMP substrates.IMPORTANCE The folding and insertion of β-barrel outer membrane proteins (OMPs) are conserved processes in mitochondria, chloroplasts, and Gram-negative bacteria. In Gram-negative bacteria, OMPs are assembled into the outer membrane (OM) by the heteropentomeric β-barrel assembly machine (BAM complex). In this study, we probe the function of the individual BAM proteins and how they coordinate assembly of a diverse family of OMPs. Furthermore, we identify a gain-of-function bamA mutant capable of assembling OMPs independently of all four other BAM proteins. This work advances our understanding of OMP assembly and sheds light on how this process is distinct in Gram-negative bacteria.

Evaluation of the β-barrel outer membrane protein VP1243 as a candidate antigen for a cross-protective vaccine against Vibrio infections

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that causes acute gastroenteritis after the consumption of contaminated food, wound infection, and seizures. Antibiotic therapy is the main method for controlling Vibrio infections, which inevitably leads to drug resistance. Therefore, a vaccine is urgently needed to avoid this problem. Outer membrane proteins (OMPs) play a pivotal role in the interaction between the host immune system and bacteria. VP1243 is an OMP of V. parahaemolyticus, and it possessed immunogenicity in our previous study. The present study found that VP1243 was widely distributed, highly conserved and possessed similar surface epitopes among the major Vibrio species. The protein stimulated a strong antibody response and induced cross-reactive immune responses in V. parahaemolyticus, V. alginolyticus and V. vulnificus. Notably, it provided 100% immune protection against lethal challenges by the three Vibrio species in mice immunized with VP1243. Efficient clearance of cells of the three Vibrio bacterial species was observed in immunized mice. These findings provide solid evidence for VP1243 as a promising candidate for the development of a versatile vaccine to protect against Vibrio infections.

Immunoinformatics Approach for Designing an Epitope-Based Peptide Vaccine against Treponema pallidum Outer Membrane Beta-Barrel Protein

Treponema pallidum is a gram negative bacteria and the main cause of syphilis which is classified as chronic inflammatory discompose antecedent transmitted sexually. Syphilis affects the central nervous system and the cardiovascular system, potentially leading to hearing or visual loss, aortic aneurism, stroke-like syndrome, dementia and paralysis. T. pallidum has the ability to stimulate adaptive immune and corresponding innate procedures in tissue and blood that might set the era for the HIV’s bidirectional transmission. This study expects a real epitope-based vaccine against β-Barrel outer membrane protein of Treponema pallidum designed by immunoinformatics approaches. The sequences were saved from NCBI and a number of prediction tests were undertaken to explore possible epitopes for B-cell, T-cell MHC class I and II. 3D structure of the most hopeful epitopes was illustrated. Two epitopes showed high binding affinity for B-cells, while five epitopes showed high binding affinity for MHCI and MHCII. The results were hopeful to formulate a vaccine with 71.88% world population coverage. We expect that these hopeful epitopes helps as a preventive formula for the disease in the future and recommend further in vitro and in vivo studies

The transmembrane supercomplex mediating the biogenesis of OMPs in Gram-negative bacteria assumes a circular conformational change upon activation.

The cell envelope of Gram-negative bacteria is composed of the inner (plasma) and the outer membrane. In the outer membrane, the outer membrane β-barrel proteins (OMPs) serve multiple functions. They are synthesized in the cytoplasm and finally inserted into the outer membrane through a critical and complex pathway facilitated by many protein factors. Recently, a new model for the biogenesis of OMPs in Gram-negative bacteria was proposed, in which a supercomplex containing multiple proteins spans the inner and outer membrane, to mediate the biogenesis of OMPs. The core part of the transmembrane supercomplex is the inner membrane protein translocon and the outer membrane β-barrel assembly machinery (BAM) complex. Some components of the supercomplex, such as the BamA subunit of the BAM complex, are essential and conserved across species. The other components, for example, the BamB subunit and the primary periplasmic chaperone SurA, are also required for the supercomplex to gain complete function and full efficiency. How BamB and SurA behave in the supercomplex, however, is less well understood. Therefore, the crosstalk between BamA, BamB and SurA was investigated mainly through invivo protein photo-cross-linking experiments and protein modeling. Moreover, theoretical structures for part of the supercomplex consisting of SurA and the BAM complex were constructed. The modeling data are consistent with the experimental results. The theoretical structures computed in this work provide a more comprehensive view of the mechanism of the supercomplex, demonstrating a circular conformational change of the supercomplex when it is active.

The gain-of-function allele bamAE470K bypasses the essential requirement for BamD in β-barrel outer membrane protein assembly

The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral β-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the β-barrel assembly machine (BAM), which is composed of one OMP-BamA-and four lipoproteins-BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation, bamA E470K , that bypasses the requirement for BamD. Although bamD::kan bamA E470K cells exhibit growth and OM barrier defects, they assemble OMPs with surprising robustness. Our results demonstrate that BamD does not play a catalytic role in OMP assembly, but rather functions to regulate the activity of BamA.

Structural insight into mitochondrial \u03b2-barrel outer membrane protein biogenesis

In mitochondria, β-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold β-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane β-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 β-barrel opens a lateral gate to accommodate its substrates.

Structural insight into the formation of lipoprotein-β-barrel complexes.

The β-barrel assembly machinery (BAM) inserts outer membrane β-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF-OMP complexes. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. RcsF is lodged deep inside the lumen of the BamA barrel, binding regions proposed to undergo an outward and lateral opening during OMP insertion. On the basis of our structural and biochemical data, we propose a push-and-pull model for RcsF export upon conformational cycling of BamA and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity.

Identification of a Compound That Inhibits the Growth of Gram-Negative Bacteria by Blocking BamA-BamD Interaction.

The demand for novel antibiotics is imperative for drug-resistant Gram-negative bacteria which causes diverse intractable infection disease in clinic. Here, a comprehensive screening was implemented to identify potential agents that disrupt the assembly of β-barrel outer-membrane proteins (OMPs) in the outer membrane (OM) of Gram-negative bacteria. The assembly of OMPs requires ubiquitous β-barrel assembly machinery (BAM). Among the five protein subunits in BAM, the interaction between BamA and BamD is essential for the function of this complex. We first established a yeast two-hybrid (Y2H) system to confirm the interaction between BamA and BamD, and then screened agents that specifically disrupt this interaction. From this screen, we identified a compound IMB-H4 that specially blocks BamA–BamD interaction and selectively inhibits the growth of Escherichia coli and other Gram-negative bacteria. Moreover, our results suggest that IMB-H4 disrupts BamA–BamD interaction by binding to BamA. Strikingly, E. coli cells having been treated with IMB-H4 showed impaired OM integrity and decreased the abundance of OMPs. Therefore, an antibacterial agent was identified successfully using Y2H system, and this compound likely blocks the assembly of OMPs by targeting BamA–BamD interaction in Gram-negative bacteria.

Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy.

The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side‐chain dynamics of the α‐helical sensory rhodopsin II and the β‐barrel outer membrane protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Despite their differing topologies, both proteins have a similar distribution of methyl‐bearing side‐chain motion that is largely independent of membrane mimetic. The methyl‐bearing side chains of both proteins are, on average, more dynamic in the ps–ns timescale than any soluble protein characterized to date. Accordingly, both proteins retain an extraordinary residual conformational entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect. Furthermore, the high conformational entropy could greatly influence the thermodynamics underlying membrane‐protein functions, including ligand binding, allostery, and signaling.

Putative β-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

Structural insights into the mechanism of a novel protein targeting pathway in Gram-negative bacteria.

Many nascent polypeptides synthesized in the cytoplasm are translocated across membranes via a specific ‘translocon’ composed of protein complexes. Recently, a novel targeting pathway for the outer membrane β‐barrel proteins (OMPs) in Gram‐negative bacteria was discovered. The cell envelope of Gram‐negative bacteria is composed of the inner (plasma) membrane (IM) and the outer membrane (OM). In this new pathway, a SecAN protein, which is mainly present in the IM as a homo‐oligomer, translocates nascent OMPs across the IM; at the same time, SecAN directly interacts with the β‐barrel assembly machinery (BAM) complex embedded within the OM. A supercomplex (containing SecAN, the BAM complex and many other proteins) spans the IM and OM, and is involved in the biogenesis of OMPs. Investigation of the function of SecAN and the supercomplex, as well as the translocation mechanism, will require elucidation of their structures. However, no such structures are available. Therefore, here, I describe the use of protein modeling to build homology models for SecAN and theoretical structures for the core‐complex composed of SecAN and the BAM complex, which is a key part of the supercomplex. The modeling data are consistent with previous experimental observations and demonstrated a conformational change of the core‐complex. I conclude by proposing mechanisms for how SecAN and the supercomplex function in the biogenesis of OMPs.

Lateral Gate Opening of BAM Complex Studied by Free Energy Calculations and the String Method

In Gram-negative bacteria, important functions such as the import of nutrients and export of virulence factors are performed by β-barrel outer membrane proteins (OMPs). These proteins are inserted and folded with the aid of the β-barrel assembly machinery (BAM). Structures of the BAM complex display two distinct conformations: a lateral-closed state and a lateral-open state. In the lateral-open state, the first and last β-strand of the BamA β-barrel separates, producing a “lateral gate” and exposing the β-barrel lumen to the outer membrane. The motion is accompanied by a significant rotation of the five polypeptide transport associated (POTRA) domains and the four accessory proteins around the barrel. Here we studied the transition between the two conformations using molecular dynamics simulations. We examined the relationship between the lateral gate opening and the rotational angle of the POTRA domains and accessory proteins using free energy calculations. We also found the minimum-energy transition pathway between the two conformations using the string method. Important contact points between the POTRA domains and the accessory proteins were found, showing how their rotation translates into the regulation of lateral gate opening.

Landscape of Eukaryotic Transmembrane Beta Barrel Proteins.

Even though in the last few years several families of eukaryotic β-barrel outer membrane proteins have been discovered, their computational characterization and their annotation in public databases are far from complete. The PFAM database includes only very few characteristic profiles for these families, and in most cases, the profile hidden Markov models (pHMMs) have been trained using prokaryotic and eukaryotic proteins together. Here, we present for the first time a comprehensive computational analysis of eukaryotic transmembrane β-barrels. Twelve characteristic pHMMs were built, based on an extensive literature search, which can discriminate eukaryotic β-barrels from other classes of proteins (globular and bacterial β-barrel ones), as well as between mitochondrial and chloroplastic ones. We built eight novel profiles for the chloroplastic β-barrel families that are not present in the PFAM database and also updated the profile for the MDM10 family (PF12519) in the PFAM database and divide the porin family (PF01459) into two separate families, namely, VDAC and TOM40.

Yeast can express and assemble bacterial secretins in the mitochondrial outer membrane.

Secretins form large multimeric pores in the outer membrane (OM) of Gram-negative bacteria. These pores are part of type II and III secretion systems (T2SS and T3SS, respectively) and are crucial for pathogenicity. Recent structural studies indicate that secretins form a structure rich in β-strands. However, little is known about the mechanism by which secretins assemble into the OM. Based on the conservation of the biogenesis of β-barrel proteins in bacteria and mitochondria, we used yeast cells as a model system to study the assembly process of secretins. To that end, we analyzed the biogenesis of PulD (T2SS), SsaC (T3SS) and InvG (T3SS) in wild type cells or in cells mutated for known mitochondrial import and assembly factors. Our results suggest that secretins can be expressed in yeast cells, where they are enriched in the mitochondrial fraction. Interestingly, deletion of mitochondrial import receptors like Tom20 and Tom70 reduces the mitochondrial association of PulD but does not affect that of InvG. SsaC shows another dependency pattern and its membrane assembly is enhanced by the absence of Tom70 and compromised in cells lacking Tom20 or the topogenesis of outer membrane β-barrel proteins (TOB) complex component, Mas37. Collectively, these findings suggest that various secretins can follow different pathways to assemble into the bacterial OM.

Outer Membrane Protein Insertion by the β-barrel Assembly Machine.

Like all outer membrane (OM) constituents, integral OM β-barrel proteins in Gram-negative bacteria are synthesized in the cytoplasm and trafficked to the OM, where they are locally assembled into the growing OM by the ubiquitous β-barrel assembly machine (Bam). While the identities and structures of all essential and accessory Bam components have been determined, the basic mechanism of Bam-assisted OM protein integration remains elusive. Here we review mechanistic analyses of OM β-barrel protein folding and Bam dynamics and summarize recent insights that inform a general model for OM protein recognition and assembly by the Bam complex.

Degp degrades a wide range of substrate proteins in Escherichia coli under stress conditions.

DegP, a periplasmic dual-functional protease and chaperone in Gram-negative bacteria, is critical for bacterial stress resistance, but the precise underlying mechanisms are not fully understood. Here, we show that the protease function of DegP is critical for Escherichia coli cells to maintain membrane integrity, particularly under heat shock conditions (42°C). Site-directed photo-cross-linking, mass spectrometry and immunoblotting analyses reveal that both periplasmic proteins (e.g. OppA and MalE) and β-barrel outer membrane proteins (OMPs) are DegP-interacting proteins and that OppA is degraded by DegP in vitro and in vivo at 42°C. In addition, OmpA and BamA, chimeric β-barrel OMPs containing a soluble periplasmic domain, are bound to DegP in both unfolded and folded forms, whereas only the unfolded forms are degradable by DegP. The presence of folded OmpA as a substrate of DegP is attributed to its periplasmic domain, which is resistant to DegP degradation and even generally protects pure β-barrel OMPs from degradation in an intra-molecular way. Furthermore, a pair of residues (R262 and V328) in the PDZ domain-1 of DegP play important roles for binding unfolded and folded β-barrel OMPs, with R262 being critical. Our study, together with earlier reports, indicates that DegP plays a critical role in protein quality control in the bacterial periplasm by degrading both periplasmic proteins and β-barrel OMPs under stress conditions and likely also by participating in the folding of chimeric β-barrel OMPs. A working model is proposed to illustrate the finely tuned functions of DegP with respect to different substrate proteins.

Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA.

The β-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane β-barrel proteins. BamA, an essential component of the complex, is itself a β-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control β-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA β-barrel. Importantly, we identify contacts between the assembling β-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze β-barrel assembly.