Biogenesis Of Outer Membrane Proteins Research Articles

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 β-barrel assembly machinery (BAM), which is a heteropentomeric complex composed of five proteins named BamA-E in E. 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 are the core BAM components and constitute 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.

Molecular Machines that Facilitate Bacterial Outer Membrane Protein Biogenesis.

Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a β-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 β-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.

ATP-independent assembly machinery of bacterial outer membranes: BAM complex structure and function set the stage for next-generation therapeutics.

Diderm bacteria employ β-barrel outer membrane proteins (OMPs) as their first line of communication with their environment. These OMPs are assembled efficiently in the asymmetric outer membrane by the β-Barrel Assembly Machinery (BAM). The multi-subunit BAM complex comprises the transmembrane OMP BamA as its functional subunit, with associated lipoproteins (e.g., BamB/C/D/E/F, RmpM) varying across phyla and performing different regulatory roles. The ability of BAM complex to recognize and fold OM β-barrels of diverse sizes, and reproducibly execute their membrane insertion, is independent of electrochemical energy. Recent atomic structures, which captured BAM-substrate complexes, show the assembly function of BamA can be tailored, with different substrate types exhibiting different folding mechanisms. Here, we highlight common and unique features of its interactome. We discuss how this conserved protein complex has evolved the ability to effectively achieve the directed assembly of diverse OMPs of wide-ranging sizes (8-36 β-stranded monomers). Additionally, we discuss how darobactin-the first natural membrane protein inhibitor of Gram-negative bacteria identified in over five decades-selectively targets and specifically inhibits BamA. We conclude by deliberating how a detailed deduction of BAM complex-associated regulation of OMP biogenesis and OM remodeling will open avenues for the identification and development of effective next-generation therapeutics against Gram-negative pathogens.

Similarly slow diffusion of BAM and SecYEG complexes in live E. coli cells observed with 3D spt-PALM

The β-barrel assembly machinery (BAM) complex is responsible for inserting outer membrane proteins (OMPs) into the Escherichia coli outer membrane. The SecYEG translocon inserts inner membrane proteins into the inner membrane and translocates both soluble proteins and nascent OMPs into the periplasm. Recent reports describe Sec possibly playing a direct role in OMP biogenesis through interactions with the soluble polypeptide transport-associated (POTRA) domains of BamA (the central OMP component of BAM). Here we probe the diffusion behavior of these protein complexes using photoactivatable super-resolution localization microscopy and single-particle tracking in live E. coli cells of BAM and SecYEG components BamA and SecE and compare them to other outer and inner membrane proteins. To accurately measure trajectories on the highly curved cell surface, three-dimensional tracking was performed using double-helix point-spread function microscopy. All proteins tested exhibit two diffusive modes characterized by “slow” and “fast” diffusion coefficients. We implement a diffusion coefficient analysis as a function of the measurement lag time to separate positional uncertainty from true mobility. The resulting true diffusion coefficients of the slow and fast modes showed a complete immobility of full-length BamA constructs in the time frame of the experiment, whereas the OMP OmpLA displayed a slow diffusion consistent with the high viscosity of the outer membrane. The periplasmic POTRA domains of BamA were found to anchor BAM to other cellular structures and render it immobile. However, deletion of individual distal POTRA domains resulted in increased mobility, suggesting that these domains are required for the full set of cellular interactions. SecE diffusion was much slower than that of the inner membrane protein PgpB and was more like OMPs and BamA. Strikingly, SecE diffused faster upon POTRA domain deletion. These results are consistent with the existence of a BAM-SecYEG trans-periplasmic assembly in live E. coli cells.

Structural basis and inhibition of outer membrane protein biogenesis in pathogenic Neisseria

Structural basis and inhibition of outer membrane protein biogenesis in pathogenic <i>Neisseria</i>

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.

Targeting BAM for Novel Therapeutics against Pathogenic Gram-Negative Bacteria.

The growing emergence of multidrug resistance in bacterial pathogens is an immediate threat to human health worldwide. Unfortunately, there has not been a matching increase in the discovery of new antibiotics to combat this alarming trend. Novel contemporary approaches aimed at antibiotic discovery against Gram-negative bacterial pathogens have expanded focus to also include essential surface-exposed receptors and protein complexes, which have classically been targeted for vaccine development. One surface-exposed protein complex that has gained recent attention is the β-barrel assembly machinery (BAM), which is conserved and essential across all Gram-negative bacteria. BAM is responsible for the biogenesis of β-barrel outer membrane proteins (β-OMPs) into the outer membrane. These β-OMPs serve essential roles for the cell including nutrient uptake, signaling, and adhesion, but can also serve as virulence factors mediating pathogenesis. The mechanism for how BAM mediates β-OMP biogenesis is known to be dynamic and complex, offering multiple modes for inhibition by small molecules and targeting by larger biologics. In this review, we introduce BAM and establish why it is a promising and exciting new therapeutic target and present recent studies reporting novel compounds and vaccines targeting BAM across various bacteria. These reports have fueled ongoing and future research on BAM and have boosted interest in BAM for its therapeutic promise in combatting multidrug resistance in Gram-negative bacterial pathogens.

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.

Generation of unfolded outer membrane protein ensembles defined by hydrodynamic properties.

Outer membrane proteins (OMPs) must exist as an unfolded ensemble while interacting with a chaperone network in the periplasm of Gram-negative bacteria. Here, we developed a method to model unfolded OMP (uOMP) conformational ensembles using the experimental properties of two well-studied OMPs. The overall sizes and shapes of the unfolded ensembles in the absence of a denaturant were experimentally defined by measuring the sedimentation coefficient as a function of urea concentration. We used these data to model a full range of unfolded conformations by parameterizing a targeted coarse-grained simulation protocol. The ensemble members were further refined by short molecular dynamics simulations to reflect proper torsion angles. The final conformational ensembles have polymer properties different from unfolded soluble and intrinsically disordered proteins and reveal inherent differences in the unfolded states that necessitate further investigation. Building these uOMP ensembles advances the understanding of OMP biogenesis and provides essential information for interpreting structures of uOMP-chaperone complexes.

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.

Characterization of the β-barrel assembly machinery in fusobacterium.

Fusobacterium nucleatum is a Gram-negative, anaerobic human oral microbiome constituent. Though a steady state commensal, this pathobiont plays a role in periodontal disease, endodontic infections, pre-term births, and colorectal cancer. F. nucleatum achieves its pathogenicity through a class of outer membrane proteins (OMPs) called adhesins. Several key adhesins present as β-barrel OMPs in the form of Type VA secretion system autotransporters or porins. The β-Barrel Assembly Machinery (BAM) complex plays a vital role in the biogenesis of β-barrel OMPs in Gram-negative bacteria. The E. coli BAM complex is composed of five proteins, OMP BamA and accessory lipoproteins BamB-E. Though there is diversity in the number of BAM complex components among Gram-negative bacteria, BamA and BamD are consistently essential for viability. However, preliminary searches of the F. nucleatum genome have revealed only the presence of BamA, the integral membrane protein of the complex, to the exclusion of any apparent periplasmic auxiliary proteins, and the structure of F. nucleatum BamA (FnBamA) remains unresolved. Thus, the objective of this study is to determine both the structure of FnBamA and to identify the accessory proteins with which it binds. We employ techniques including X-ray crystallography and cryo-electron microscopy (EM) to accomplish the structural aims of this proposal and have obtained the first-ever structure of FnBamA using cryo-EM. Pull-down assays coupled with proteomics elucidate the identity and nature of proteins accessory to BamA. This research is innovative in its exploration of membrane protein biology in F. nucleatum through examining BAM, a difficult system unstudied in this challenging anaerobic organism. We expect to advance the mitigation of F. nucleatum pathogenesis in the oral microbiome and beyond by detecting the binding partners and solving the structure of FnBamA.

BAM folds OMPs using a budding mechanism.

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

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.

Deep learning-driven insights into super protein complexes for outer membrane protein biogenesis in bacteria

To reach their final destinations, outer membrane proteins (OMPs) of gram-negative bacteria undertake an eventful journey beginning in the cytosol. Multiple molecular machines, chaperones, proteases, and other enzymes facilitate the translocation and assembly of OMPs. These helpers usually associate, often transiently, forming large protein assemblies. They are not well understood due to experimental challenges in capturing and characterizing protein-protein interactions (PPIs), especially transient ones. Using AF2Complex, we introduce a high-throughput, deep learning pipeline to identify PPIs within the Escherichia coli cell envelope and apply it to several proteins from an OMP biogenesis pathway. Among the top confident hits obtained from screening ~1500 envelope proteins, we find not only expected interactions but also unexpected ones with profound implications. Subsequently, we predict atomic structures for these protein complexes. These structures, typically of high confidence, explain experimental observations and lead to mechanistic hypotheses for how a chaperone assists a nascent, precursor OMP emerging from a translocon, how another chaperone prevents it from aggregating and docks to a β-barrel assembly port, and how a protease performs quality control. This work presents a general strategy for investigating biological pathways by using structural insights gained from deep learning-based predictions.

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.

A network of cytosolic (co)chaperones promotes the biogenesis of mitochondrial signal-anchored outer membrane proteins.

Signal-anchored (SA) proteins are anchored into the mitochondrial outer membrane (OM) via a single transmembrane segment at their N-terminus while the bulk of the proteins is facing the cytosol. These proteins are encoded by nuclear DNA, translated on cytosolic ribosomes, and are then targeted to the organelle and inserted into its OM by import factors. Recently, research on the insertion mechanisms of these proteins into the mitochondrial OM have gained a lot of attention. In contrast, the early cytosolic steps of their biogenesis are unresolved. Using various proteins from this category and a broad set of in vivo, in organello, and in vitro assays, we reconstituted the early steps of their biogenesis. We identified a subset of molecular (co)chaperones that interact with newly synthesized SA proteins, namely, Hsp70 and Hsp90 chaperones and co-chaperones from the Hsp40 family like Ydj1 and Sis1. These interactions were mediated by the hydrophobic transmembrane segments of the SA proteins. We further demonstrate that interfering with these interactions inhibits the biogenesis of SA proteins to a various extent. Finally, we could demonstrate direct interaction of peptides corresponding to the transmembrane segments of SA proteins with the (co)chaperones and reconstitute in vitro the transfer of such peptides from the Hsp70 chaperone to the mitochondrial Tom70 receptor. Collectively, this study unravels an array of cytosolic chaperones and mitochondrial import factors that facilitates the targeting and membrane integration of mitochondrial SA proteins.

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.

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed.

Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates

Periplasmic chaperones 17-kilodalton protein (Skp) and survival factor A (SurA) are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA associations with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy-entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multifaceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.

Outer membrane protein biogenesis in pathogenic Neisseria

Neisseria gonorrhoeae (Ngo), is an obligate human pathogen and the causative agent of the disease gonorrhea. If left untreated, gonorrhea can lead to serious health issues including ectopic pregnancy and infertility. In addition to unsuccessful vaccine development, Ngo has rapidly become resistant to almost all classes of antibiotics, which contribute to over 500,000 drug resistant cases in year in the U.S. Due to this widespread resistance, the current recommended treatment option has become limited to a single drug of last resort: ceftriaxone.