Folding Of Outer Membrane Proteins Research Articles

Outer membrane protein assembly mediated by BAM-SurA complexes

The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded β-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis.

Protein–lipid charge interactions control the folding of outer membrane proteins into asymmetric membranes

Biological membranes consist of two leaflets of phospholipid molecules that form a bilayer, each leaflet comprising a distinct lipid composition. This asymmetry is created and maintained in vivo by dedicated biochemical pathways, but difficulties in creating stable asymmetric membranes in vitro have restricted our understanding of how bilayer asymmetry modulates the folding, stability and function of membrane proteins. In this study, we used cyclodextrin-mediated lipid exchange to generate liposomes with asymmetric bilayers and characterize the stability and folding kinetics of two bacterial outer membrane proteins (OMPs), OmpA and BamA. We found that excess negative charge in the outer leaflet of a liposome impedes their insertion and folding, while excess negative charge in the inner leaflet accelerates their folding relative to symmetric liposomes with the same membrane composition. Using molecular dynamics, mutational analysis and bioinformatics, we identified a positively charged patch critical for folding and stability. These results rationalize the well-known ‘positive-outside’ rule of OMPs and suggest insights into the mechanisms that drive OMP folding and assembly in vitro and in vivo.

Drug Binding to BamA Targets Its Lateral Gate.

BamA, the core component of the β-barrel assembly machinery (BAM) complex, is an outer-membrane protein (OMP) in Gram-negative bacteria. Its function is to insert and fold substrate OMPs into the outer membrane (OM). Evidence suggests that BamA follows the asymmetric hybrid-barrel model where the first and last strands of BamA separate, a process known as lateral gate opening, to allow nascent substrate OMP β-strands to sequentially insert and fold through β-augmentation. Recently, multiple lead compounds that interfere with BamA's function have been identified. We modeled and then docked one of these compounds into either the extracellular loops of BamA or the open lateral gate. With the compound docked in the loops, we found that the lateral gate remains closed during 5 μs molecular dynamics simulations. The same compound when docked in the open lateral gate stays bound to the β16 strand of BamA during the simulation, which would prevent substrate OMP folding. In addition, we simulated mutants of BamA that are resistant to one or more of the identified lead compounds. In these simulations, we observed a differing degree and/or frequency of opening of BamA's lateral gate compared to BamA-apo, suggesting that the mutations grant resistance by altering the dynamics at the gate. We conclude that the compounds act by inhibiting BamA lateral gate opening and/or binding of substrate, thus preventing subsequent OMP folding and insertion.

BamE directly interacts with BamA and BamD coordinating their functions.

The β-barrel assembly machinery (Bam) complex facilitates the assembly of outer membrane proteins (OMPs) in gram-negative bacteria. The Bam complex is conserved and essential for bacterial viability and consists of five subunits, BamA-E. BamA is the transmembrane component, and its β-barrel domain opens laterally to allow folding and insertion of incoming OMPs. The remaining components are regulatory, among which only BamD is essential. Previous studies suggested that BamB regulates BamA directly, while BamE and BamC serve as BamD regulators. However, specific molecular details of their functions remain unknown. Our previous research demonstrated that BamE plays a specialized role in assembling the complex between the lipoprotein RcsF and its OMP partners, required for the Regulator of Capsule Synthesis (Rcs) stress response. Here, we used RcsF/OmpA as a model substrate to investigate BamE function. Our results challenge the current view that BamE only serves as a BamD regulator. We show that BamE also directly interacts with BamA. BamE interaction with both BamA and BamD is important for function. Our genetic and biochemical analysis shows that BamE stabilizes the Bam complex and promotes bidirectional signaling interaction between BamA and BamD. This BamE function becomes essential when direct BamA/BamD communication is impeded.

β-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 β-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.

FkpA enhances membrane protein folding using an extensive interaction surface.

Outer membrane protein (OMP) biogenesis in gram-negative bacteria is managed by a network of periplasmic chaperones that includes SurA, Skp, and FkpA. These chaperones bind unfolded OMPs (uOMPs) in dynamic conformational ensembles to suppress aggregation, facilitate diffusion across the periplasm, and enhance folding. FkpA primarily responds to heat-shock stress, but its mechanism is comparatively understudied. To determine FkpA chaperone function in the context of OMP folding, we monitored the folding of three OMPs and found that FkpA, unlike other periplasmic chaperones, increases the folded yield but decreases the folding rate of OMPs. The results indicate that FkpA behaves as a chaperone and not as a folding catalyst to influence the OMP folding trajectory. Consistent with the folding assay results, FkpA binds all three uOMPs as determined by sedimentation velocity (SV) and photo-crosslinking experiments. We determine the binding affinity between FkpA and uOmpA171 by globally fitting SV titrations and find it to be intermediate between the known affinities of Skp and SurA for uOMP clients. Notably, complex formation steeply depends on the urea concentration, suggesting an extensive binding interface. Initial characterizations of the complex using photo-crosslinking indicate that the binding interface spans the entire FkpA molecule. In contrast to prior findings, folding and binding experiments performed using subdomain constructs of FkpA demonstrate that the full-length chaperone is required for full activity. Together these results support that FkpA has a distinct and direct effect on OMP folding that it achieves by utilizing an extensive chaperone-client interface to tightly bind clients.

FkpA enhances outer membrane protein folding using an extensive interaction surface.

Outer membrane protein (OMP) biogenesis in gram-negative bacteria is managed by a network of periplasmic chaperones that includes SurA, Skp, and FkpA. These chaperones bind unfolded OMPs (uOMPs) in dynamic conformational ensembles to suppress uOMP aggregation, facilitate diffusion across the periplasm, and enhance OMP folding. FkpA primarily responds to heat-shock stress, but its mechanism is comparatively understudied. To determine FkpA chaperone function, we monitored the folding of a cognate client uOmpA171 and found that FkpA increases the folded uOmpA171population but also slows the folding rate, dual functions distinct from the other periplasmic chaperones. The results indicate that FkpA behaves as a chaperone and not as a folding catalyst to directly influence the uOmpA171 folding trajectory. We determine the binding affinity between FkpA and uOmpA171 by globally fitting sedimentation velocity titrations and found it to be intermediate between the known affinities of Skp and SurA for uOMP clients. Notably, complex formation steeply depends on the urea concentration, suggestive of an extensive binding interface. Initial characterizations of the complex using photo-crosslinking indicates that the binding interface spans the inner surfaces of the entire FkpA molecule. In contrast to prior findings, folding and binding experiments performed using subdomain constructs of FkpA demonstrate that the full-length chaperone is required for full activity. Together these results support that FkpA has a distinct and direct effect on uOMP folding and that it achieves this by utilizing an extensive chaperone-client interface.

The delivery complex in outer membrane protein biogenesis: How the SurA chaperone interacts with the BAM enzyme.

SurA is the major chaperone of unfolded outer membrane proteins (OMPs) in the periplasm of E. coli. Its function has not been fully resolved but current literature suggests roles in binding OMPs as they emerge from the inner membrane, preventing their aggregation, and maintaining them in a folding competent state. Less well studied is how OMPs can be delivered to the BAM complex via SurA diffusing in the periplasm. We use AlphaFold models as a starting point to explore the ‘delivery complex’ between SurA and BAM. Through the solution of cryoEM-derived structures, in vitro biophysics, and in vivo functional assays we can build a mechanistic model that incorporates chaperone delivery into the current BAM-assisted OMP folding paradigm.

Outer membrane protein folding mechanism: How does an OMP reach the active site of the BAM complex?

Outer membrane proteins (OMPs) are folded into the outer membrane of E. coli by the BAM complex. Substrate OMPs are transported through the periplasm as unfolded proteins and delivered to the BAM complex. Recent cryoEM structures of the BAM complex folding substrates have focused on late stages of the mechanism when the substrate has engaged with the BamA lateral gate and successive beta-strands are formed until the OMP is released as a beta-barrel. It is unclear how substrate OMPs arrive and engage at the BamA barrel in the correct orientation. AlphaFold was used to guide the design of crosslinks to trap BAM-chaperone-substrate complexes in vivo. CryoEM ternary complexes aid the understanding of how a substrate is delivered from a chaperone to the active site of the BAM complex.

Single-molecule approaches reveal outer membrane protein biogenesis dynamics.

Outer membrane proteins (OMPs) maintain the viability of Gram-negative bacteria by functioning as receptors, transporters, ion channels, lipases, and porins. Folding and assembly of OMPs involves synchronized action of chaperones and multi-protein machineries which escort the highly hydrophobic polypeptides to their target outer membrane in a folding competent state. Previous studies have identified proteins and their involvement along the OMP biogenesis pathway. Yet, the mechanisms of action and the intriguing ability of all these molecular machines to work without the typical cellular energy source of ATP, but solely based on thermodynamic principles, are still not well understood. Here, we highlight how different single-molecule studies can shed additional light on the mechanisms and kinetics of OMP biogenesis.

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.

The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli.

The outer membrane of Gram-negative bacteria acts as an additional diffusion barrier for solutes and nutrients. It is perforated by outer membrane proteins (OMPs) that function most often as diffusion pores, but sometimes also as parts of larger cellular transport complexes, structural components of the cell wall, or even as enzymes. These OMPs often have large loops that protrude into the extracellular environment, which have promise for biotechnological applications and as therapeutic targets. Thus, understanding how modifications to these loops affect OMP stability and folding is critical for their efficient application. In this work, the small outer membrane protein OmpX was used as a model system to quantify the effects of loop insertions on OMP folding and stability. The insertions were varied according to both hydrophobicity and size, and their effects were determined by assaying folding into detergent micelles in vitro by SDS-PAGE and in vivo by isolating the outer membrane of cells expressing the constructs. The different insertions were also examined in molecular dynamics simulations to resolve how they affect OmpX dynamics in its native outer membrane. The results indicate that folding of OMPs is affected by both the insert length and by its hydrophobic character. Small insertions sometimes even improved the folding efficiency of OmpX, while large hydrophilic inserts reduced it. All the constructs that were found to fold in vitro could also do so in their native environment. One construct that could not fold in vitro was transported to the OM in vivo, but remained unfolded. Our results will help to improve the design and efficiency of recombinant OMPs used for surface display.

Dynamic interplay between the periplasmic chaperone SurA and the BAM complex in outer membrane protein folding.

Correct folding of outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria depends on delivery of unfolded OMPs to the β-barrel assembly machinery (BAM). How unfolded substrates are presented to BAM remains elusive, but the major OMP chaperone SurA is proposed to play a key role. Here, we have used hydrogen deuterium exchange mass spectrometry (HDX-MS), crosslinking, in vitro folding and binding assays and computational modelling to show that the core domain of SurA and one of its two PPIase domains are key to the SurA-BAM interaction and are required for maximal catalysis of OMP folding. We reveal that binding causes changes in BAM and SurA conformation and/or dynamics distal to the sites of binding, including at the BamA β1-β16 seam. We propose a model for OMP biogenesis in which SurA plays a crucial role in OMP delivery and primes BAM to accept substrates for folding.

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding

Transmembrane β barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the β barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model β barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "β signal" motif of EspP to correctly orient β strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated β sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that β sheets progressively fold toward BamA to form a β barrel. Along with invivo experiments that tracked β barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate β barrel folding.

Efficient reconstitution of beta-barrel proteins for single-molecule experiments

The outer membrane of Gram-negative bacteria is populated with vital beta-barrel transmembrane proteins, so-called outer-membrane proteins (OMPs). These proteins fold and insert into the outer membrane from the periplasm in absence of any known energy source. Unlike the widely examined process of alpha-helical transmembrane protein folding, the complex steps involved in OMP biogenesis remain obscure. However, in recent years, the development of single-molecule techniques (such as force spectroscopy and single-molecule FRET) has allowed researchers to markedly advance the observation of OMP folding and insertion.

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

The biogenesis of integral β-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 β-barrel assembly machine (Bam) complex. We find that β-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 β-barrel folding by directly mediating the displacement and degradation of assembly-compromised OMP substrates from the Bam complex.

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.

The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding

The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.

Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors.

The development of new antibiotics is particularly problematic in Gram-negative bacteria due to the presence of the outer membrane (OM), which serves as a permeability barrier. Recently, the β-barrel assembly machine (BAM), located in the OM and responsible for β-barrel type OM protein (OMP) assembly, has been validated as a novel target for antibiotics. Here, we identified potential BAM complex inhibitors using a screening approach that reports on cell envelope σE and Rcs stress in Escherichia coli. Screening a library consisting of 316 953 compounds yielded five compounds that induced σE and Rcs stress responses, while not inducing the intracellular heat-shock response. Two of the five compounds (compounds 2 and 14) showed the characteristics of known BAM complex inhibitors: synergy with OMP biogenesis mutants, decrease in the abundance of various OMPs, and loss of OM integrity. Importantly, compound 2 also inhibited BAM-dependent OMP folding in an in vitro refolding assay using purified BAM complex reconstituted in proteoliposomes.

Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress.

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.