Insertion Of Outer Membrane Proteins Research Articles

Identification of an Essential LolD-Like Protein in Helicobacter pylori.

The localization of lipoprotein (Lol) system is used by Gram-negative bacteria to export lipoproteins to the outer membrane. Lol proteins and models of how Lol transfers lipoproteins from the inner to the outer membrane have been extensively characterized in the model organism Escherichia coli, but in numerous bacterial species, lipoprotein synthesis and export pathways deviate from the E. coli paradigm. For example, in the human gastric bacterium Helicobacter pylori, a homolog of the E. coli outer membrane component LolB is not found, E. coli LolC and LolE correspond to a single inner membrane component (LolF), and a homolog of the E. coli cytoplasmic ATPase LolD has not been identified. In the present study, we sought to identify a LolD-like protein in H. pylori. We used affinity-purification mass spectrometry to identify interaction partners of the H. pylori ATP-binding cassette (ABC) family permease LolF and identified the ABC family ATP-binding protein HP0179 as its interaction partner. We engineered H. pylori to conditionally express HP0179 and showed that HP0179 and its conserved ATP binding and ATP hydrolysis motifs are essential for H. pylori growth. We then performed affinity purification-mass spectrometry using HP0179 as the bait and identified LolF as its interaction partner. These results indicate that H. pylori HP0179 is a LolD-like protein and provide a more complete understanding of lipoprotein localization processes in H. pylori, a bacterium in which the Lol system deviates from the E. coli paradigm. IMPORTANCE Lipoproteins are critical in Gram-negative-bacteria for cell surface assembly of LPS, insertion of outer membrane proteins, and sensing envelope stress. Lipoproteins also contribute to bacterial pathogenesis. For many of these functions, lipoproteins must localize to the Gram-negative outer membrane. Transporting lipoproteins to the outer membrane involves the Lol sorting pathway. Detailed analyses of the Lol pathway have been performed in the model organism Escherichia coli, but many bacteria utilize altered components or are missing essential components of the E. coli Lol pathway. Identifying a LolD-like protein in Helicobacter pylori is important to better understand the Lol pathway in diverse bacterial classes. This becomes particularly relevant as lipoprotein localization is targeted for antimicrobial development.

A bacterial secretosome for regulated envelope biogenesis and quality control?

The Gram-negative bacterial envelope is the first line of defence against environmental stress and antibiotics. Therefore, its biogenesis is of considerable fundamental interest, as well as a challenge to address the growing problem of antimicrobial resistance. All bacterial proteins are synthesised in the cytosol, so inner- and outer-membrane proteins, and periplasmic residents have to be transported to their final destinations via specialised protein machinery. The Sec translocon, a ubiquitous integral inner-membrane (IM) complex, is key to this process as the major gateway for protein transit from the cytosol to the cell envelope; this can be achieved during their translation, or afterwards. Proteins need to be directed into the inner-membrane (usually co-translational), otherwise SecA utilises ATP and the proton-motive-force (PMF) to drive proteins across the membrane post-translationally. These proteins are then picked up by chaperones for folding in the periplasm, or delivered to the β-barrel assembly machinery (BAM) for incorporation into the outer-membrane. The core hetero-trimeric SecYEG-complex forms the hub for an extensive network of interactions that regulate protein delivery and quality control. Here, we conduct a biochemical exploration of this 'secretosome' -a very large, versatile and inter-changeable assembly with the Sec-translocon at its core; featuring interactions that facilitate secretion (SecDF), inner- and outer-membrane protein insertion (respectively, YidC and BAM), protein folding and quality control (e.g. PpiD, YfgM and FtsH). We propose the dynamic interplay amongst these, and other factors, act to ensure efficient envelope biogenesis, regulated to accommodate the requirements of cell elongation and division. We believe this organisation is critical for cell wall biogenesis and remodelling and thus its perturbation could be a means for the development of anti-microbials.

Modeling intermediates of BamA folding an outer membrane protein

BamA, the core component of the β-barrel assembly machinery complex, is an integral outer-membrane protein (OMP) in Gram-negative bacteria that catalyzes the folding and insertion of OMPs. A key feature of BamA relevant to its function is a lateral gate between its first and last β-strands. Opening of this lateral gate is one of the first steps in the asymmetric-hybrid-barrel model of BamA function. In this study, multiple hybrid-barrel folding intermediates of BamA and a substrate OMP, EspP, were constructed and simulated to better understand the model’s physical consequences. The hybrid-barrel intermediates consisted of the BamA β-barrel and its POTRA5 domain and either one, two, three, four, five, or six β-hairpins of EspP. The simulation results support an asymmetric-hybrid-barrel model in which the BamA N-terminal β-strand forms stronger interactions with the substrate OMP than the C-terminal β-strand. A consistent “B”-shaped conformation of the final folding intermediate was observed, and the shape of the substrate β-barrel within the hybrid matched the shape of the fully folded substrate. Upon further investigation, inward-facing glycines were found at sharp bends within the hybrid and fully folded β-barrels. Together, the data suggest an influence of sequence on shape of the substrate barrel throughout the OMP folding process and of the fully folded OMP.

Comparing the dynamics of the BAM complex between two species

Outer membrane proteins (OMPs) in Gram-negative bacteria are transmembrane β-barrel proteins that are involved in important biological activities such as nutrient transport, signal transduction and the export of virulence factors. While it’s known that the protein complex responsible for assembly and insertion of OMPs into the outer membrane is the β-barrel assembly machinery (BAM), the detailed mechanisms for OMP insertion by BAM are only recently coming into focus. Nonetheless, to date, the only complete high-resolution structures of BAM are from Escherichia coli, leaving open the question of the degree of conservation of mechanisms across species.

The best defense is a cheap offense? How Gram-negative bacteria build their outer membranes and secrete toxins without typical sources of energy

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 OMPs utilize the β-barrel assembly machinery (BAM) to insert and fold into the outer membrane. A leading model of OMP insertion by BAM proposes that β-strands of the nascent OMP are threaded into the open lateral gate of the core BAM component, BamA, forming hydrogen bonds with the exposed strands, known as the “hybrid-barrel” model.

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.

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.

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.

The Peripheral Outer Membrane Protein BamB from E. coli Binds in a Random Surface Orientation to Lipid Membranes. a Site-Directed Fluorescence Study

The β-barrel assembly machinery (BAM) complex of Gram-negative bacteria like E. coli is required for folding and insertion of outer membrane proteins (OMPs) into the outer membrane. In E. coli, the complex consists of the subunits BamA, B, C, D, and E. While BamA contains a transmembrane domain, the other subunits are peripheral membrane proteins. Deletion of bamB leads to reduced concentrations of folded OMPs, like OmpA or LamB. Here we have investigated the interface of BamB with lipid bilayers. Amino acid residues distributed over the entire surface of BamB were selected for replacement by a cysteine to prepare a range of single-cysteine mutants of BamB. Correct structure of the isolated mutants was confirmed by circular dichroism spectroscopy and mutants were labeled with fluorescent dye IANBD at the cysteine. Fluorescence spectroscopy and fluorescence quenching by spin-labeled lipids demonstrated that the entire surface of BamB interacts with the lipid bilayer, indicating a random surface orientation of BamB. The N-terminal lipid modification of BamB was not required for membrane binding.An increased binding of BamB was observed for membranes composed of PG only, likely because of electrostatic interactions. CD spectroscopy indicated that PG alters the structure of BamB, converting about ∼8 % of the residues that form β-strands to α-helical conformation. These observations may have implications for the function of BamB to improve folding of OMPs into the outer membrane.

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.

Tic22 from Anabaena sp. PCC 7120 with holdase function involved in outer membrane protein biogenesis shuttles between plasma membrane and Omp85.

β-barrel-shaped outer membrane proteins (OMPs) ensure regulated exchange of molecules across the cell-wall of Gram-negative bacteria. They are synthesized in the cytoplasm and translocated across the plasma membrane via the SEC translocon. In the periplasm, several proteins participate in the transfer of OMPs to the outer membrane-localized complex catalyzing their insertion. This process has been described in detail for proteobacteria and some molecular components are conserved in cyanobacteria. For example, Omp85 proteins that catalyze the insertion of OMPs into the outer membrane exist in cyanobacteria as well. In turn, SurA and Skp involved in OMP transfer from plasma membrane to Omp85 in E. coli are likely replaced by Tic22 in cyanobacteria. We describe that anaTic22 functions as periplasmic holdase for OMPs in Anabaena sp. PCC 7120 and provide evidence for the process of substrate delivery to anaOmp85. AnaTic22 binds to the plasma membrane with specificity for phosphatidylglycerol and monogalactosyldiacylglycerol. Substrate recognition induces membrane dissociation and interaction with the N-terminal POTRA domain of Omp85. This leads to substrate release by the interaction with a proline-rich domain and the first POTRA domain of Omp85. The order of events during OMP transfer from plasma membrane to Omp85 in cyanobacteria is discussed.

TIC236 links the outer and inner membrane translocons of the chloroplast.

The two-membrane envelope is a defining feature of chloroplasts. Chloroplasts evolved from a Gram-negative cyanobacterial endosymbiont. During evolution, genes of the endosymbiont have been transferred to the host nuclear genome. Most chloroplast proteins are synthesized in the cytosol as higher-molecular-mass preproteins with an N-terminal transit peptide. Preproteins are transported into chloroplasts by the TOC and TIC (translocons at the outer- and inner-envelope membranes of chloroplasts, respectively) machineries1,2, but how TOC and TIC are assembled together is unknown. Here we report the identification of the TIC component TIC236; TIC236 is an integral inner-membrane protein that projects a 230-kDa domain into the intermembrane space, which binds directly to the outer-membrane channel TOC75. The knockout mutation of TIC236 is embryonically lethal. In TIC236-knockdown mutants, a smaller amount of the inner-membrane channel TIC20 was associated with TOC75; the amount of TOC-TIC supercomplexes was also reduced. This resulted in a reduced import rate into the stroma, though outer-membrane protein insertion was unaffected. The size and the essential nature of TIC236 indicate that-unlike in mitochondria, in which the outer- and inner-membrane translocons exist as separate complexes and a supercomplex is only transiently assembled during preprotein translocation3,4-a long and stable protein bridge in the intermembrane space is required for protein translocation into chloroplasts. Furthermore, TIC236 and TOC75 are homologues of bacterial inner-membrane TamB5 and outer-membrane BamA, respectively. Our evolutionary analyses show that, similar to TOC75, TIC236 is preserved only in plants and has co-evolved with TOC75 throughout the plant lineage. This suggests that the backbone of the chloroplast protein-import machinery evolved from the bacterial TamB-BamA protein-secretion system.

C-terminal kink formation is required for lateral gating in BamA

In Gram-negative bacteria, the outer membrane contains primarily β-barrel transmembrane proteins and lipoproteins. The insertion and assembly of β-barrel outer-membrane proteins (OMPs) is mediated by the β-barrel assembly machinery (BAM) complex, the core component of which is the 16-stranded transmembrane β-barrel BamA. Recent studies have indicated a possible role played by the seam between the first and last β-barrel strands of BamA in the OMP insertion process through lateral gating and a destabilized membrane region. In this study, we have determined the stability and dynamics of the lateral gate through over 12.5 μs of equilibrium simulations and 4 μs of free-energy calculations. From the equilibrium simulations, we have identified a persistent kink in the C-terminal strand and observed spontaneous lateral-gate separation in a mimic of the native bacterial outer membrane. Free-energy calculations of lateral gate opening revealed a significantly lower barrier to opening in the C-terminal kinked conformation; mutagenesis experiments confirm the relevance of C-terminal kinking to BamA structure and function.

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "β-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively but often requires non-physiological conditions and time scales and is strongly influenced by properties of the lipid bilayer, including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the β-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel-phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA, and another model protein (OmpG) folded at significantly different rates and that an α-helix embedded inside the EspP β-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding.

BamA β16C strand and periplasmic turns are critical for outer membrane protein insertion and assembly

Outer membrane (OM) β-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins (OMPs) are inserted and assembled into the OM by OMP85 family proteins. In Escherichia coli, the β-barrel assembly machinery (BAM) contains four lipoproteins such as BamB, BamC, BamD and BamE, and one OMP BamA, forming a 'top hat'-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel β1C-β6C scissor-like movement that promote the unfolded OMP insertion without using ATP. Here, we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the β16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique β16C strand and the periplasmic turns of BamA are important for the outer membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA β1C-β6C, triggering the OMP insertion. These results are important for understanding the OMP insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts.

Selective pressure for rapid membrane integration constrains the sequence of bacterial outer membrane proteins.

Almost all bacterial outer membrane proteins (OMPs) contain a β barrel domain that serves as a membrane anchor, but the assembly and quality control of these proteins are poorly understood. Here, we show that the introduction of a single lipid-facing arginine residue near the middle of the β barrel of the Escherichia coli OMPs OmpLA and EspP creates an energy barrier that impedes membrane insertion. Although several unintegrated OmpLA mutants remained insertion-competent, they were slowly degraded by the periplasmic protease DegP. Two EspP mutants were also gradually degraded by DegP but were toxic because they first bound to the Bam complex, an essential heteroligomer that catalyzes the membrane insertion of OMPs. Interestingly, another EspP mutant likewise formed a prolonged, deleterious interaction with the Bam complex but was protected from degradation and eventually inserted into the membrane in a native conformation. The different types of interactions between the EspP mutants and the Bam complex that we observed may correspond to distinct stages in OMP assembly. Our results show that sequences that significantly delay assembly are disfavored not only because unintegrated OMPs are subjected to degradation, but also because OMPs that assemble slowly can form dominant-negative interactions with the Bam complex.

Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding

The biogenesis of outer-membrane proteins (OMPs) in gram-negative bacteria involves delivery by periplasmic chaperones to the β-barrel assembly machinery (BAM), which catalyzes OMP insertion into the outer membrane. Here, we examine the effects of membrane thickness, the Escherichia coli periplasmic chaperones Skp and SurA, and BamA, the central subunit of the BAM complex, on the folding kinetics of a model OMP (tOmpA) using fluorescence spectroscopy, native mass spectrometry, and molecular dynamics simulations. We show that prefolded BamA promotes the release of tOmpA from Skp despite the nM affinity of the Skp:tOmpA complex. This activity is located in the BamA β-barrel domain, but is greater when full-length BamA is present, indicating that both the β-barrel and polypeptide transport-associated (POTRA) domains are required for maximal activity. By contrast, SurA is unable to release tOmpA from Skp, providing direct evidence against a sequential chaperone model. By varying lipid acyl chain length in synthetic liposomes we show that BamA has a greater catalytic effect on tOmpA folding in thicker bilayers, suggesting that BAM catalysis involves lowering of the kinetic barrier imposed by the hydrophobic thickness of the membrane. Consistent with this, molecular dynamics simulations reveal that increases in membrane thinning/disorder by the transmembrane domain of BamA is greatest in thicker bilayers. Finally, we demonstrate that cross-linking of the BamA barrel does not affect tOmpA folding kinetics in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes, suggesting that lateral gating of the BamA barrel and/or hybrid barrel formation is not required, at least for the assembly of a small 8-stranded OMP in vitro.

Extreme Dynamics in the BamA β-Barrel Seam.

BamA is an essential component of the β-barrel assembly machine (BAM) that is responsible for insertion and folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. BamA is an OMP itself, and its β-barrel transmembrane domain is thought to catalyze OMP insertion and folding, although the molecular mechanism remains poorly understood. Crystal structures of BamA and complementary molecular dynamics simulations have shown that its β-barrel seam (the interface between the first and last barrel strands) is destabilized. This has led to mechanistic models in which the BamA barrel seam functions as a lateral gate that opens and successively accepts β-hairpins from a nascent OMP such that a nascent barrel can bud from BamA. Consistent with this model, disulfide locking of the BamA barrel seam is lethal in Escherichia coli. Here we show that disulfide locking of the BamA barrel has no effect on its ability to catalyze folding of a model OMP into liposomes. However, disulfide trapping experiments indicate that the BamA barrel is highly dynamic in the liposome membranes, with the β-strands at the barrel seam undergoing "register sliding" by more than 14 Å both up and down the membrane. Remarkably, these extreme dynamics were also observed in the BamA barrel in the context of the native E. coli outer membrane. These results are consistent with a model in which the BamA barrel dynamics induce defects in the outer membrane that facilitate insertion of nascent OMPs.

Folding outer membrane proteins independently of the β-barrel assembly machinery: an assembly pathway for multimeric complexes?

Since the discovery of the essential role of the β-barrel assembly machinery (BAM) for the membrane insertion of outer membrane proteins (OMPs) that are unrelated in sequence, members of this universally conserved family dominate discussions on OMP assembly in bacteria, mitochondria and chloroplasts. However, several multimeric bacterial OMPs assemble independently of the catalyzing BAM-component BamA. Recent progress on this alternative pathway is reviewed here, and a model for BAM-independent assembly for multimeric OMPs is proposed in which monomer delivery to the membrane and stable prepore formation are key steps towards productive membrane insertion.

Structural basis of outer membrane protein insertion by the BAM complex

All Gram-negative bacteria, mitochondria and chloroplasts have outer membrane proteins (OMPs) that perform many fundamental biological processes. The OMPs in Gram-negative bacteria are inserted and folded into the outer membrane by the β-barrel assembly machinery (BAM). The mechanism involved is poorly understood, owing to the absence of a structure of the entire BAM complex. Here we report two crystal structures of the Escherichia coli BAM complex in two distinct states: an inward-open state and a lateral-open state. Our structures reveal that the five polypeptide transport-associated domains of BamA form a ring architecture with four associated lipoproteins, BamB-BamE, in the periplasm. Our structural, functional studies and molecular dynamics simulations indicate that these subunits rotate with respect to the integral membrane β-barrel of BamA to induce movement of the β-strands of the barrel and promote insertion of the nascent OMP.