Molecular Machines that Facilitate Bacterial Outer Membrane Protein Biogenesis.

Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a ß-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the ß-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.

Programmed Secretion Arrest and Receptor-Triggered Toxin Export during Antibacterial Contact-Dependent Growth Inhibition.

Contact-dependent growth inhibition (CDI) entails receptor-mediated delivery of CdiA-derived toxins into Gram-negative target bacteria. Using electron cryotomography, we show that each CdiA effector protein forms a filament extending ∼33 nm from the cell surface. Remarkably, the extracellular filament represents only the N-terminal half of the effector. A programmed secretion arrest sequesters the C-terminal half of CdiA, including the toxin domain, in the periplasm prior to target-cell recognition. Upon binding receptor, CdiA secretion resumes, and the periplasmic FHA-2 domain is transferred to the target-cell outer membrane. The C-terminal toxin region of CdiA then penetrates into the target-cell periplasm, where it is cleaved for subsequent translocation into the cytoplasm. Our findings suggest that the FHA-2 domain assembles into a transmembrane conduit for toxin transport into the periplasm of target bacteria. We propose that receptor-triggered secretion ensures that FHA-2 export is closely coordinated with integration into the target-cell outer membrane. VIDEO ABSTRACT.

Function of the Omp85 Superfamily of Outer Membrane Protein Assembly Factors and Polypeptide Transporters.

The Omp85 protein superfamily is found in the outer membrane (OM) of all gram-negative bacteria and eukaryotic organelles of bacterial origin. Members of the family catalyze both the membrane insertion of ß-barrel proteins and the translocation of proteins across the OM. Although the mechanism(s) by which these proteins function is unclear, striking new insights have emerged from recent biochemical and structural studies. In this review we discuss the entire Omp85 superfamily but focus on the function of the best-studied member, BamA, which is an essential and highly conserved component of the bacterial barrel assembly machinery (BAM). Because BamA has multiple functions that overlap with those of other Omp85 proteins, it is likely the prototypical member of the Omp85 superfamily. Furthermore, BamA has become a protein of great interest because of the recent discovery of small-molecule inhibitors that potentially represent an important new class of antibiotics.

General features of transmembrane beta barrels from a large database.

Large datasets contribute new insights to subjects formerly investigated by exemplars. We used coevolution data to create a large, high-quality database of transmembrane ß-barrels (TMBB). By applying simple feature detection on generated evolutionary contact maps, our method (IsItABarrel) achieves 95.88% balanced accuracy when discriminating among protein classes. Moreover, comparison with IsItABarrel revealed a high rate of false positives in previous TMBB algorithms. In addition to being more accurate than previous datasets, our database (available online) contains 1,938,936 bacterial TMBB proteins from 38 phyla, respectively, 17 and 2.2 times larger than the previous sets TMBB-DB and OMPdb. We anticipate that due to its quality and size, the database will serve as a useful resource where high-quality TMBB sequence data are required. We found that TMBBs can be divided into 11 types, three of which have not been previously reported. We find tremendous variance in proteome percentage among TMBB-containing organisms with some using 6.79% of their proteome for TMBBs and others using as little as 0.27% of their proteome. The distribution of the lengths of the TMBBs is suggestive of previously hypothesized duplication events. In addition, we find that the C-terminal ß-signal varies among different classes of bacteria though its consensus sequence is LGLGYRF. However, this ß-signal is only characteristic of prototypical TMBBs. The ten non-prototypical barrel types have other C-terminal motifs, and it remains to be determined if these alternative motifs facilitate TMBB insertion or perform any other signaling function.

A minimum functional form of the Escherichia coli BAM complex constituted by BamADE assembles outer membrane proteins in vitro.

The biogenesis of outer membrane proteins is mediated by the ß-barrel assembly machinery (BAM), which is a heteropentomeric complex composed of five proteins named BamA-E in Escherichia coli. Despite great progress in the BAM structural analysis, the molecular details of BAM-mediated processes as well as the exact function of each BAM component during OMP assembly are still not fully understood. To enable a distinguishment of the function of each BAM component, it is the aim of the present work to examine and identify the effective minimum form of the E. coli BAM complex by use of a well-defined reconstitution strategy based on a previously developed versatile assay. Our data demonstrate that BamADE is the core BAM component and constitutes a minimum functional form for OMP assembly in E. coli, which can be stimulated by BamB and BamC. While BamB and BamC have a redundant function based on the minimum form, both together seem to cooperate with each other to substitute for the function of the missing BamD or BamE. Moreover, the BamAE470K mutant also requires the function of BamD and BamE to assemble OMPs in vitro, which vice verse suggests that BamADE are the effective minimum functional form of the E. coli BAM complex.

Membrane insertases at a glance.

Protein translocases, such as the bacterial SecY complex, the Sec61 complex of the endoplasmic reticulum (ER) and the mitochondrial translocases, facilitate the transport of proteins across membranes. In addition, they catalyze the insertion of integral membrane proteins into the lipid bilayer. Several membrane insertases cooperate with these translocases, thereby promoting the topogenesis, folding and assembly of membrane proteins. Oxa1 and BamA family members serve as core components in the two major classes of membrane insertases. They facilitate the integration of proteins with α-helical transmembrane domains and of ß-barrel proteins into lipid bilayers, respectively. Members of the Oxa1 family were initially found in the internal membranes of bacteria, mitochondria and chloroplasts. Recent studies, however, also identified several Oxa1-type insertases in the ER, where they serve as catalytically active core subunits in the ER membrane protein complex (EMC), the guided entry of tail-anchored (GET) and the GET- and EMC-like (GEL) complex. The outer membrane of bacteria, mitochondria and chloroplasts contain ß-barrel proteins, which are inserted by members of the BamA family. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of these different types of membrane insertases and discuss their function.

The sacrificial adaptor protein Skp functions to remove stalled substrates from the ß-barrel assembly machine.

The biogenesis of integral ß-barrel outer membrane proteins (OMPs) in gram-negative bacteria requires transport by molecular chaperones across the aqueous periplasmic space. Owing in part to the extensive functional redundancy within the periplasmic chaperone network, specific roles for molecular chaperones in OMP quality control and assembly have remained largely elusive. Here, by deliberately perturbing the OMP assembly process through use of multiple folding-defective substrates, we have identified a role for the periplasmic chaperone Skp in ensuring efficient folding of OMPs by the ß-barrel assembly machine (Bam) complex. We find that ß-barrel substrates that fail to integrate into the membrane in a timely manner are removed from the Bam complex by Skp, thereby allowing for clearance of stalled Bam-OMP complexes. Following the displacement of OMPs from the assembly machinery, Skp subsequently serves as a sacrificial adaptor protein to directly facilitate the degradation of defective OMP substrates by the periplasmic protease DegP. We conclude that Skp acts to ensure efficient ß-barrel folding by directly mediating the displacement and degradation of assembly-compromised OMP substrates from the Bam complex.

Benzylic C(sp3 )-H Bond Oxidation with Ketone Selectivity by a Cobalt(IV)-Oxo Embedded in a ß-Barrel Protein.

Artificial metalloenzymes have emerged as biohybrid catalysts that allow to combine the reactivity of a metal catalyst with the flexibility of protein scaffolds. This work reports the artificial metalloenzymes based on the ß-barrel protein nitrobindin NB4, in which a cofactor [CoII X(Me3 TACD-Mal)]+ X- (X=Cl, Br; Me3 TACD=N,N' ,N''-trimethyl-1,4,7,10-tetraazacyclododecane, Mal=CH2 CH2 CH2 NC4 H2 O2 ) was covalently anchored via a Michael addition reaction. These biohybrid catalysts showed higher efficiency than the free cobalt complexes for the oxidation of benzylic C(sp3 )-H bonds in aqueous media. Using commercially available oxone (2KHSO5 ⋅ KHSO4 ⋅ K2 SO4 ) as oxidant, a total turnover number of up to 220 and 97 % ketone selectivity were achieved for tetralin. As catalytically active intermediate, a mononuclear terminal cobalt(IV)-oxo species [Co(IV)=O]2+ was generated by reacting the cobalt(II) cofactor with oxone in aqueous solution and characterized by ESI-TOF MS.

Licochalcone A inhibits the assembly function of ß-barrel assembly machinery in Escherichia coli.

Antimicrobial resistance (AMR) crisis urges the development of new antibiotics. In the present work, we for the first time used bio-affinity ultrafiltration combined with HPLC-MS (UF-HPLC-MS) to examine the interaction between the outer membrane ß-barrel proteins and natural products. Our results showed that natural product licochalcone A from licorice interacts with BamA and BamD with the enrichment factor of 6.38 ± 1.46 and 4.80 ± 1.23, respectively. The interaction was further confirmed by use of biacore analysis, which demonstrated that the Kd value between BamA/D and licochalcone was 6.63/28.27 µM, suggesting a good affinity. To examine the effect of licochalcone A on BamA/D function, the developed versatile in vitro reconstitution assay was used and the results showed that 128 µg/mL licochalcone A could reduce the outer membrane protein A integration efficiency to 20%. Although licochalcone A alone can not inhibit the growth of E. coli, but it can affect the membrane permeability, suggesting that licochalcone A holds the potential to be used as a sensitizer to combat AMR.

Anatomy of a selectively coassembled ß-sheet peptide nanofiber.

Peptide self-assembly, wherein molecule A associates with other A molecules to form fibrillar ß-sheet structures, is common in nature and widely used to fabricate synthetic biomaterials. Selective coassembly of peptide pairs A and B with complementary partial charges is gaining interest due to its potential for expanding the form and function of biomaterials that can be realized. It has been hypothesized that charge-complementary peptides organize into alternating ABAB-type arrangements within assembled ß-sheets, but no direct molecular-level evidence exists to support this interpretation. We report a computational and experimental approach to characterize molecular-level organization of the established peptide pair, CATCH. Discontinuous molecular dynamics simulations predict that CATCH(+) and CATCH(-) peptides coassemble but do not self-assemble. Two-layer ß-sheet amyloid structures predominate, but off-pathway ß-barrel oligomers are also predicted. At low concentration, transmission electron microscopy and dynamic light scattering identified nonfibrillar ∼20-nm oligomers, while at high concentrations elongated fibers predominated. Thioflavin T fluorimetry estimates rapid and near-stoichiometric coassembly of CATCH(+) and CATCH(-) at concentrations ≥100 µM. Natural abundance 13C NMR and isotope-edited Fourier transform infrared spectroscopy indicate that CATCH(+) and CATCH(-) coassemble into two-component nanofibers instead of self-sorting. However, 13C-13C dipolar recoupling solid-state NMR measurements also identify nonnegligible AA and BB interactions among a majority of AB pairs. Collectively, these results demonstrate that strictly alternating arrangements of ß-strands predominate in coassembled CATCH structures, but deviations from perfect alternation occur. Off-pathway ß-barrel oligomers are also suggested to occur in coassembled ß-strand peptide systems.

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

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a ß-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.

ß-Barrel Assembly Machinery (BAM) Complex as Novel Antibacterial Drug Target.

The outer membrane of Gram-negative bacteria is closely related to the pathogenicity and drug resistance of bacteria. Outer membrane proteins (OMPs) are a class of proteins with important biological functions on the outer membrane. The ß-barrel assembly machinery (BAM) complex plays a key role in OMP biogenesis, which ensures that the OMP is inserted into the outer membrane in a correct folding manner and performs nutrient uptake, antibiotic resistance, cell adhesion, cell signaling, and maintenance of membrane stability and other functions. The BAM complex is highly conserved among Gram-negative bacteria. The abnormality of the BAM complex will lead to the obstruction of OMP folding, affect the function of the outer membrane, and eventually lead to bacterial death. In view of the important role of the BAM complex in OMP biogenesis, the BAM complex has become an attractive target for the development of new antibacterial drugs against Gram-negative bacteria. Here, we summarize the structure and function of the BAM complex and review the latest research progress of antibacterial drugs targeting BAM in order to provide a new perspective for the development of antibiotics.

Voltage-activated complexation of α-synuclein with three diverse ß-barrel channels: VDAC, MspA, and α-hemolysin.

Voltage-activated complexation is the process by which a transmembrane potential drives complex formation between a membrane-embedded channel and a soluble or membrane-peripheral target protein. Metabolite and calcium flux across the mitochondrial outer membrane was shown to be regulated by voltage-activated complexation of the voltage-dependent anion channel (VDAC) and either dimeric tubulin or α-synuclein (αSyn). However, the roles played by VDAC's characteristic attributes-its anion selectivity and voltage gating behavior-have remained unclear. Here, we compare in vitro measurements of voltage-activated complexation of αSyn with three well-characterized ß-barrel channels-VDAC, MspA, and α-hemolysin-that differ widely in their organism of origin, structure, geometry, charge density distribution, and voltage gating behavior. The voltage dependences of the complexation dynamics for the different channels are observed to differ quantitatively but have similar qualitative features. In each case, energy landscape modeling describes the complexation dynamics in a manner consistent with the known properties of the individual channels, while voltage gating does not appear to play a role. The reaction free energy landscapes thus calculated reveal a non-trivial dependence of the αSyn/channel complex stability on the surface density of αSyn.

The assembly of ß-barrel membrane proteins by BAM and SAM.

Gram-negative bacteria, mitochondria, and chloroplasts all possess an outer membrane populated with a host of ß-barrel outer-membrane proteins (ßOMPs). These ßOMPs play crucial roles in maintaining viability of their hosts, and therefore, it is essential to understand the biogenesis of this class of membrane proteins. In recent years, significant structural and functional advancements have been made toward elucidating this process, which is mediated by the ß-barrel assembly machinery (BAM) in Gram-negative bacteria, and by the sorting and assembly machinery (SAM) in mitochondria. Structures of both BAM and SAM have now been reported, allowing a comparison and dissection of the two machineries, with other studies reporting on functional aspects of each. Together, these new insights provide compelling support for the proposed budding mechanism, where each nascent ßOMP forms a hybrid-barrel intermediate with BAM/SAM in route to its biogenesis into the membrane. Here, we will review these recent studies and highlight their contributions toward understanding ßOMP biogenesis in Gram-negative bacteria and in mitochondria. We will also weigh the evidence supporting each of the two leading mechanistic models for how BAM/SAM function, and offer an outlook on future studies within the field.

The Thermodynamic Stability of Membrane Proteins in Micelles and Lipid Bilayers Investigated with the Ferrichrom Receptor FhuA.

Extraction of integral membrane proteins into detergents for structural and functional studies often leads to a strong loss in protein stability. The impact of the lipid bilayer on the thermodynamic stability of an integral membrane protein in comparison to its solubilized form in detergent was examined and compared for FhuA from Escherichia coli and for a mutant, FhuAΔ5-160, lacking the N-terminal cork domain. Urea-induced unfolding was monitored by fluorescence spectroscopy to determine the effective free energies [Formula: see text] of unfolding. To obtain enthalpic and entropic contributions of unfolding of FhuA, [Formula: see text] were determined at various temperatures. When solubilized in LDAO detergent, wt-FhuA and FhuAΔ5-160 unfolded in a single step. The 155-residue cork domain stabilized wt-FhuA by [Formula: see text]~ 40 kJ/mol. Reconstituted into lipid bilayers, wt-FhuA unfolded in two steps, while FhuAΔ5-160 unfolded in a single step, indicating an uncoupled unfolding of the cork domain. For FhuAΔ5-160 at 35 °C, [Formula: see text] increased from ~ 5 kJ/mol in LDAO micelles to about ~ 20 kJ/mol in lipid bilayers, while the temperature of unfolding increased from TM ~ 49 °C in LDAO micelles to TM ~ 75 °C in lipid bilayers. Enthalpies [Formula: see text]were much larger than free energies [Formula: see text], for FhuAΔ5-160 and for wt-FhuA, and compensated by a large gain of entropy upon unfolding. The gain in conformational entropy is expected to be similar for unfolding of FhuA from micelles or bilayers. The strongly increased TM and [Formula: see text] observed for the lipid bilayer-reconstituted FhuA in comparison to the LDAO-solubilized forms, therefore, very likely arise from a much-increased solvation entropy of FhuA in bilayers.

Mosaic Evolution of Beta-Barrel-Porin-Encoding Genes in Escherichia coli.

Bacterial porin-encoding genes are often found under positive selection. Local recombination has also been identified in a few of them to facilitate bacterial rapid adaptation, although it remains unknown whether it is a common evolutionary mechanism for the porins or outer membrane proteins in Gram-negative bacteria. In this study, we investigated the beta-barrel (ß-barrel) porin-encoding genes in Escherichia coli that were reported under positive Darwinian selection. Besides fhuA that was found with ingenic local recombination previously, we identified four other genes, i.e., lamB, ompA, ompC, and ompF, all showing the similar mosaic evolution patterns. Comparative analysis of the protein sequences disclosed a list of highly variable regions in each family, which are mostly located in the convex of extracellular loops and coinciding with the binding sites of bacteriophages. For each of the porin families, mosaic recombination leads to unique combinations of the variable regions with different sequence patterns, generating diverse protein groups. Structural modeling indicated a conserved global topology among the different porins, with the extracellular surface varying a lot due to individual or combinatorial variable regions. The conservation of global tertiary structure would ensure the channel activity, while the wide diversity of variable regions may represent selection to avoid the invasion of phages, antibiotics or immune surveillance factors. Our study identified multiple bacterial porin genes with mosaic evolution. We hypothesize that this could be generalized strategy for outer membrane proteins to both maintain normal life processes and evade the attack of unfavored factors rapidly. IMPORTANCE Microevolution studies can disclose more elaborate evolutionary mechanisms of genes, appearing especially important for genes with multifaceted function such as those encoding outer membrane proteins. However, in most cases, the gene is considered as a whole unit, and the evolutionary patterns are disclosed. Here, we report that multiple bacterial porin proteins follow mosaic evolution, with local ingenic recombination combined with spontaneous mutations based on positive Darwinian selection, and conservation for most structural regions. This could represent a common mechanism for bacterial outer membrane proteins. The variable regions within each porin family showed large coincidence with the binding sites of bacteriophages, antibiotics, and immune factors and therefore would represent effective targets for the development of new antibacterial agents or vaccines.

Overexpression of the Bam Complex Improves the Production of Chlamydia trachomatis MOMP in the E. coli Outer Membrane.

A licensed Chlamydia trachomatis (Ct) vaccine is not yet available. Recombinant Chlamydia trachomatis major outer membrane protein (Ct-MOMP), the most abundant constituent of the chlamydial outer membrane complex, is considered the most attractive candidate for subunit-based vaccine formulations. Unfortunately, Ct-MOMP is difficult to express in its native structure in the E. coli outer membrane (OM). Here, by co-expression of the Bam complex, we improved the expression and localization of recombinant Ct-MOMP in the E. coli OM. Under these conditions, recombinant Ct-MOMP appeared to assemble into a ß-barrel conformation and express domains at the cell surface indicative of correct folding. The data indicate that limited availability of the Bam complex can be a bottleneck for the production of heterologous OM vaccine antigens, information that is also relevant for strategies aimed at producing recombinant OMV-based vaccines.

New Insights into the Chloroplast Outer Membrane Proteome and Associated Targeting Pathways.

Plastids are a dynamic class of organelle in plant cells that arose from an ancient cyanobacterial endosymbiont. Over the course of evolution, most genes encoding plastid proteins were transferred to the nuclear genome. In parallel, eukaryotic cells evolved a series of targeting pathways and complex proteinaceous machinery at the plastid surface to direct these proteins back to their target organelle. Chloroplasts are the most well-characterized plastids, responsible for photosynthesis and other important metabolic functions. The biogenesis and function of chloroplasts rely heavily on the fidelity of intracellular protein trafficking pathways. Therefore, understanding these pathways and their regulation is essential. Furthermore, the chloroplast outer membrane proteome remains relatively uncharted territory in our understanding of protein targeting. Many key players in the cytosol, receptors at the organelle surface, and insertases that facilitate insertion into the chloroplast outer membrane remain elusive for this group of proteins. In this review, we summarize recent advances in the understanding of well-characterized chloroplast outer membrane protein targeting pathways as well as provide new insights into novel targeting signals and pathways more recently identified using a bioinformatic approach. As a result of our analyses, we expand the known number of chloroplast outer membrane proteins from 117 to 138.

Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria.

ß-Barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and morphology, nutrient acquisition, colonization and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs must fold and insert into a crowded and asymmetric OM that lacks much freely accessible lipid. This feat is accomplished in the absence of an external energy source and is thought to be driven by the high thermodynamic stability of folded OMPs in the OM. With such a stable fold, the challenge that bacteria face in assembling OMPs into the OM is how to overcome the initial energy barrier of membrane insertion. In this review, we highlight the roles of the lipid environment and the OM in modulating the OMP-folding landscape and discuss the factors that guide folding in vitro and in vivo We particularly focus on the composition, architecture, and physical properties of the OM and how an understanding of the folding properties of OMPs in vitro can help explain the challenges they encounter during folding in vivo Current models of OMP biogenesis in the cellular environment are still in flux, but the stakes for improving the accuracy of these models are high. OMP folding is an essential process in all Gram-negative bacteria, and considering the looming crisis of widespread microbial drug resistance it is an attractive target. To bring down this vital OMP-supported barrier to antibiotics, we must first understand how bacterial cells build it.

TtOmp85, a single Omp85 member protein functions as a ß-barrel protein insertase and an autotransporter translocase without any accessory proteins.

The biogenesis of outer membrane proteins requires the function of ß-barrel assembly machinery (BAM), whose function is highly conserved while its composition is variable. The Escherichia coli BAM is composed of five subunits, while Thermus thermophilus seems to contain a single BAM protein, named TtOmp85. To search for the primitive form of a functional BAM, we investigated and compared the function of TtOmp85 and E. coli BAM by use of a reconstitution assay that examines the integration of OmpA and BamA from E. coli and TtoA from T. thermophilus, as well as the translocation of the E. coli Ag43. Our results show that a single TtOmp85 protein can substitute for the collective function of the five subunits constituting E. coli BAM.