Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.

Peptidoglycan maturation controls outer membrane protein assembly.

Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial for the maintenance of cellular integrity and enable survival in challenging environments1-5. The function of the outer membrane is dependent on outer membrane proteins (OMPs), which are inserted into the membrane by the ß-barrel assembly machine6,7 (BAM). Growing Escherichia coli cells segregate old OMPs towards the poles by a process known as binary partitioning, the basis of which is unknown8. Here we demonstrate that peptidoglycan underpins the spatiotemporal organization of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little effect on its activity, leading to preferential insertion of OMPs at division sites. The synchronization of OMP biogenesis with cell wall growth results in the binary partitioning of OMPs as cells divide. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation, a potential vulnerability that could be exploited in future antibiotic design.

The vaccinia chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion.

Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.

An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system.

The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.

The lipoprotein DolP affects cell separation in Escherichia coli, but not as an upstream regulator of NlpD.

Bacterial amidases are essential to split the shared envelope of adjunct daughter cells to allow cell separation. Their activity needs to be precisely controlled to prevent cell lysis. In Escherichia coli, amidase activity is controlled by three regulatory proteins NlpD, EnvC and ActS. However, recent studies linked the outer membrane lipoprotein DolP (formerly YraP) as a potential upstream regulator of NlpD. In this study we explored this link in further detail. To our surprise DolP did not modulate amidase activity in vitro and was unable to interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. Next, we excluded the hypothesis that ΔdolP phenocopied ΔnlpD in a range of envelope stresses. However, morphological analysis of double deletion mutants of amidases (AmiA, AmiB AmiC) and amidase regulators with dolP revealed that ΔamiAΔdolP and ΔenvCΔdolP mutants display longer chain length compared to their parental strains indicating a role for DolP in cell division. Overall, we present evidence that DolP does not affect NlpD function in vitro, implying that DolP is not an upstream regulator of NlpD. However, DolP may impact daughter cell separation by interacting directly with AmiA or AmiC, or by a yet undiscovered mechanism.

Surface-tethered planar membranes containing the ß-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 ß-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.

Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation.

The solubilisation of membrane proteins (MPs) necessitates the overlap of two contradictory events; the extraction of MPs from their native lipid membranes and their subsequent stabilisation in aqueous environments. Whilst the current myriad of membrane mimetic systems provide a range of modus operandi, there are no golden rules for selecting the optimal pipeline for solubilisation of a specific MP hence a miscellaneous approach must be employed balancing both solubilisation efficiency and protein stability. In recent years, numerous diverse lipid membrane mimetic systems have been developed, expanding the pool of available solubilisation strategies. This review provides an overview of recent developments in the membrane mimetic field, with particular emphasis placed upon detergents, polymer-based nanodiscs and amphipols, highlighting the latest reagents to enter the toolbox of MP research.

YraP Contributes to Cell Envelope Integrity and Virulence of Salmonella enterica Serovar Typhimurium.

Mutations in σE-regulated lipoproteins have previously been shown to impact bacterial viability under conditions of stress and during in vivo infection. YraP is conserved across a number of Gram-negative pathogens, including Neisseria meningitidis, where the homolog is a component of the Bexsero meningococcal group B vaccine. Investigations using laboratory-adapted Escherichia coli K-12 have shown that yraP mutants have elevated sensitivity to a range of compounds, including detergents and normally ineffective antibiotics. In this study, we investigate the role of the outer membrane lipoprotein YraP in the pathogenesis of Salmonella enterica serovar Typhimurium. We show that mutations in S Typhimurium yraP result in a defective outer membrane barrier with elevated sensitivity to a range of compounds. This defect is associated with attenuated virulence in an oral infection model and during the early stages of systemic infection. We show that this attenuation is not a result of defects in lipopolysaccharide and O-antigen synthesis, changes in outer membrane protein levels, or the ability to adhere to and invade eukaryotic cell lines in vitro.

Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates.

The isolated apical domain of GroEL consisting of residues 191-345 (known as "minichaperone") binds and assists the folding of a wide variety of client proteins without GroES and ATP, but the mechanism of its action is still unknown. In order to probe into the matter, we have examined minichaperone-mediated folding of a large aggregation prone protein Maltodextrin-glucosidase (MalZ). The key objective was to identify whether MalZ exists free in solution, or remains bound to, or cycling on and off the minichaperone during the refolding process. When GroES was introduced during refolding process, production of the native MalZ was inhibited. We also observed the same findings with a trap mutant of GroEL, which stably captures a predominantly non-native MalZ released from minichaperone during refolding process, but does not release it. Tryptophan and ANS fluorescence measurements indicated that refolded MalZ has the same structure as the native MalZ, but that its structure when bound to minichaperone is different. Surface plasmon resonance measurements provide an estimate for the equilibrium dissociation constant KD for the MalZ-minichaperone complex of 0.21 ±â€¯0.04 µM, which are significantly higher than for most GroEL clients. This showed that minichaperone interacts loosely with MalZ to allow the protein to change its conformation and fold while bound during the refolding process. These observations suggest that the minichaperone works by carrying out repeated cycles of binding aggregation-prone protein MalZ in a relatively compact conformation and in a partially folded but active state, and releasing them to attempt to fold in solution.

Characterization of a Putative Receptor Binding Surface on Skint-1, a Critical Determinant of Dendritic Epidermal T Cell Selection.

Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection.

A novel pathway for outer membrane protein biogenesis in Gram-negative bacteria.

The understanding of the biogenesis of the outer membrane of Gram-negative bacteria is of critical importance due to the emergence of bacteria that are becoming resistant to available antibiotics. A problem that is most serious for Gram-negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram-negative bacterial outer membrane is therefore critical to developing new antimicrobial agents, as this membrane makes direct contact with the external milieu, and the proteins present within this membrane are the instruments of microbial warfare, playing key roles in microbial pathogenesis, virulence and multidrug resistance. To date, a single outer membrane complex has been identified as essential for the folding and insertion of proteins into the outer membrane, this is the ß-barrel assembly machine (BAM) complex, which in some cases is supplemented by the Translocation and Assembly Module (TAM). In this issue of Molecular Microbiology, Dunstan et al. have identified a novel pathway for the insertion of a subset of integral membrane proteins into the Gram-negative outer membrane that is independent of the BAM complex and TAM.

Cross-species chimeras reveal BamA POTRA and ß-barrel domains must be fine-tuned for efficient OMP insertion.

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of ß-barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded ß-barrel precursors via the five polypeptide transport-associated (POTRA) domains at its N-terminus. The C-terminus of BamA folds into a ß-barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram-negative bacteria and appear to function in a species-specific manner. Here we investigate the nature of this species-specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the ß-barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the ß-barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the ß-barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.

Evolutionary history of copy-number-variable locus for the low-affinity Fcγ receptor: mutation rate, autoimmune disease, and the legacy of helminth infection.

Both sequence variation and copy-number variation (CNV) of the genes encoding receptors for immunoglobulin G (Fcγ receptors) have been genetically and functionally associated with a number of autoimmune diseases. However, the molecular nature and evolutionary context of this variation is unknown. Here, we describe the structure of the CNV, estimate its mutation rate and diversity, and place it in the context of the known functional alloantigen variation of these genes. Deletion of Fcγ receptor IIIB, associated with systemic lupus erythematosus, is a result of independent nonallelic homologous recombination events with a frequency of approximately 0.1%. We also show that pathogen diversity, in particular helminth diversity, has played a critical role in shaping the functional variation at these genes both between mammalian species and between human populations. Positively selected amino acids are involved in the interaction with IgG and include some amino acids that are known polymorphic alloantigens in humans. This supports a genetic contribution to the hygiene hypothesis, which states that past evolution in the context of helminth diversity has left humans with an array of susceptibility alleles for autoimmune disease in the context of a helminth-free environment. This approach shows the link between pathogens and autoimmune disease at the genetic level and provides a strategy for interrogating the genetic variation underlying autoimmune-disease risk and infectious-disease susceptibility.

Detergent-free purification of ABC (ATP-binding-cassette) transporters.

ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.

Distance tuneable integral membrane protein containing floating bilayers via in situ directed self-assembly.

Model membranes allow for structural and biophysical studies on membrane biochemistry at the molecular level, albeit on systems of reduced complexity which can limit biological accuracy. Floating supported bilayers offer a means of producing planar lipid membrane models not adhered to a surface, which allows for improved accuracy compared to other model membranes. Here we communicate the incorporation of an integral membrane protein complex, the multidomain ß-barrel assembly machinery (Bam), into our recently developed in situ self-assembled floating supported bilayers. Using neutron reflectometry and quartz crystal microbalance measurements we show this sample system can be fabricated using a two-step self-assembly process. We then demonstrate the complexity of the model membrane and tuneability of the membrane-to-surface distance using changes in the salt concentration of the bulk solution. Results demonstrate an easily fabricated, biologically accurate and tuneable membrane assay system which can be utilized for studies on integral membrane proteins within their native lipid matrix.

Structure and function of BamE within the outer membrane and the ß-barrel assembly machine.

Insertion of folded proteins into the outer membrane of Gram-negative bacteria is mediated by the essential ß-barrel assembly machine (Bam). Here, we report the native structure and mechanism of a core component of this complex, BamE, and show that it is exclusively monomeric in its native environment of the periplasm, but is able to adopt a distinct dimeric conformation in the cytoplasm. BamE is shown to bind specifically to phosphatidylglycerol, and comprehensive mutagenesis and interaction studies have mapped key determinants for complex binding, outer membrane integrity and cell viability, as well as revealing the role of BamE within the Bam complex.

A generalised module for the selective extracellular accumulation of recombinant proteins.

BACKGROUND: It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. RESULTS: Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. CONCLUSIONS: The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications.

The essential ß-barrel assembly machinery complex components BamD and BamA are required for autotransporter biogenesis.

Autotransporter biogenesis is dependent upon BamA, a central component of the ß-barrel assembly machinery (BAM) complex. In this report, we detail the role of the other BAM components (BamB-E). We identify the importance of BamD in autotransporter biogenesis and show that BamB, BamC, and BamE are not required.

Surfactant-free purification of membrane proteins with intact native membrane environment.

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10 nm disc-like structure ideally suited to purification and further biochemical study.

Adsorption of a styrene maleic acid (SMA) copolymer-stabilized phospholipid nanodisc on a solid-supported planar lipid bilayer.

Over recent years, there has been a rapid development of membrane-mimetic systems to encapsulate and stabilize planar segments of phospholipid bilayers in solution. One such system has been the use of amphipathic copolymers to solubilize lipid bilayers into nanodiscs. The attractiveness of this system, in part, stems from the capability of these polymers to solubilize membrane proteins directly from the host cell membrane. The assumption has been that the native lipid annulus remains intact, with nanodiscs providing a snapshot of the lipid environment. Recent studies have provided evidence that phospholipids can exchange from the nanodiscs with either lipids at interfaces, or with other nanodiscs in bulk solution. Here we investigate kinetics of lipid exchange between three recently studied polymer-stabilized nanodiscs and supported lipid bilayers at the silicon-water interface. We show that lipid and polymer exchange occurs in all nanodiscs tested, although the rate and extent differs between different nanodisc types. Furthermore, we observe adsorption of nanodiscs to the supported lipid bilayer for one nanodisc system which used a polymer made using reversible addition-fragmentation chain transfer polymerization. These results have important implications in applications of polymer-stabilized nanodiscs, such as in the fabrication of solid-supported films containing membrane proteins.