Β-barrel Outer Membrane Proteins Research Articles

LD-transpeptidation is crucial for fitness and polar growth in Agrobacterium tumefaciens.

Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species also employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane β-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.

The Assembly of the Inverse Autotransporter Protein YeeJ is Driven by its C-terminal β-strand

Autotransporter proteins are bacterial outer membrane proteins that display passenger domains with various functions through a β-barrel shaped translocation domain. YeeJ is an autotransporter protein from E. coli MG1655. In contrast to most other autotransporter proteins, its passenger domain is located at the C-terminus of the translocation domain. Due to this inverted domain organization, YeeJ belongs to autotransporter proteins of type Ve. To investigate the assembly of YeeJ, the fluorescence of a heterologous mCherry passenger domain was measured to quantify its assembly. Based on AlphaFold2 models of 119 sequences similar to YeeJ, a sequence conservation logo for the β1- and the β12-strand of type Ve autotransporter proteins was generated. Then, the effect of mutations in these strands on the assembly of YeeJ were analyzed. Mutations of the N-terminal aromatic amino acid of the β1-strand did not affect the assembly of the translocation domain and the display of the passenger domain. Likewise, exchange of the β1-strand with the β3-strand did not impair the assembly of the autotransporter fusion protein. Mutation of the C-terminal aromatic amino acid of the β12-strand strongly impaired surface display of the mCherry passenger domain. This amino acid has been shown before as an essential feature of the β-signals of classical autotransporter proteins and outer membrane β-barrel proteins in general. We therefore propose that the β12-strand of YeeJ acts as its β-signal and that the assembly of the YeeJ β-barrel is driven by its C-terminal β-strand, like in most other autotransporter proteins, despite its inverted domain organization.

Determining the functional role of the Gluconobacter oxydans GOX1969 protein as a BamB homolog.

Acetic acid bacteria are used in many industrial processes such as the production of vinegar, vitamin C, the antidiabetic drug miglitol, and various artificial flavorings. These industrially important reactions are primarily carried out by an arsenal of periplasmic-facing membrane-bound dehydrogenases that incompletely oxidize their substrates and shuttle electrons directly into the respiratory chain. Among these dehydrogenases, GOX1969 in Gluconobacter oxydans was predicted to be a pyrroloquinoline quinone-dependent dehydrogenase of unknown function. However, after multiple analysis by a number of labs, no dehydrogenase activity has been detected. Reanalysis of GOX1969 sequence and structure reveals similarities to Escherichia coli BamB, which functions as a subunit of the β-barrel assembly machinery complex that is responsible for the assembly of β-barrel outer membrane proteins in Gram-negative bacteria. To test if the physiological function of GOX1969 is similar to BamB in E. coli, we introduced the gox1969 gene into an E. coli ∆bamB mutant. Growth deficiencies in the ∆bamB mutant were restored when gox1969 was expressed on the plasmid pGox1969. Furthermore, increased membrane permeability conferred by bamB deletion was restored upon gox1969 expression, which suggests a direct link between GOX1969 and a role in maintaining outer membrane stability. Together, this evidence strongly suggests that GOX1969 is functionally acting as a BamB in G. oxydans. As such, functional information on uncharacterized genes will provide new insights that will allow for more accurate modeling of acetic acid bacterial metabolism and further efforts to design rational strains for industrial use.IMPORTANCEGluconobacter oxydans is an industrially important member of the acetic acid bacteria. Experimental characterization of putative genes is necessary to identify targets for further engineering of rational acetic acid bacteria strains that can be used in the production of vitamin C, antidiabetic compounds, artificial flavorings, or novel compounds. In this study, we have identified an undefined dehydrogenase GOX1969 with no known substrate and defined structural similarities to outer membrane biogenesis protein BamB in E. coli K12. Furthermore, we demonstrate that GOX1969 is capable of complementing bamB knockout phenotypes in E. coli K12. Taken together, these findings enhance our understanding of G. oxydans physiology and expand the list of potential targets for future industrial strain design.

The Name Is Barrel, β-Barrel.

β-barrels are a class of membrane proteins made up of a cylindrical, anti-parallel β-sheet with a hydrophobic exterior and a hydrophilic interior. The majority of proteins found in the outer membranes (OMs) of Gram-negative bacteria, mitochondria, and chloroplasts are β-barrel outer membrane proteins (OMPs). β-barrel OMPs have a diverse repertoire of functions, including nutrient transport, secretion, bacterial virulence, and enzymatic activity. Here, we discuss the broad functional classes of β-barrel OMPs, how they are folded into the membrane, and the future of β-barrel OMP research and its applications.

Stress-Based Screening for Compounds That Inhibit β-Barrel Outer Membrane Protein Assembly in Gram-Negative Bacteria.

Biogenesis of the outer membrane (OM) of Gram-negative bacteria involves two processes essential for growth, that is, the insertion of β-barrel outer membrane proteins (OMPs) by the Bam complex and the assembly of the LPS-containing outer leaflet of the OM by the LptD/E complex from the Lpt pathway. These processes have only recently gained attention as targets for antimicrobial drugs. Our laboratory has developed a simple screening tool to identify compounds that target processes that disrupt the biogenesis of the cell envelope, among which the activity of the Bam complex. The tool is based on the observation that such a disruption triggers cell envelope stress response systems, such as the σE, Rcs, and Cpx responses. In essence, specific stress-responsive promoters are fused to a gene encoding a bright fluorescent protein to serve as a panel of easy-to-monitor stress reporter plasmids. Using these plasmids, compounds triggering these stress systems and, therefore, putatively disrupting the biogenesis of the cell envelope can be identified by the nature and kinetics of the induced stress responses. We describe here the use of the stress reporter plasmids in high-throughput phenotypic screening using multi-well plates.

Characterization of β-Barrel Outer Membrane Proteins and Their Interactions with Chaperones by Chemical-Crosslinking Mass Spectrometry.

Chemical crosslinking-mass spectrometry (XL-MS) is an established tool that can be used to study the architecture and dynamics of proteins and protein assemblies. Here the application of XL-MS to study outer membrane proteins (OMPs) and their interactions with periplasmic chaperones is described, to inform on the molecular mechanisms underpinning OMP assembly. XL-MS data are especially powerful when used to complement high-resolution structural data, data from structural prediction or to drive molecular modeling of proteins and protein assemblies. The approach described here could be applied to the study of any protein assembly (including other membrane proteins).

SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains.

The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane β-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2-4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic β-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate β-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.

Multiple Rounds of In Vivo Random Mutagenesis and Selection in Vibrio natriegens Result in Substantial Increases in REE Binding Capacity.

Rare earth elements (REE) are essential ingredients in many modern technologies, yet their purification remains either environmentally harmful or economically unviable. Adsorption, or biosorption, of REE onto bacterial cell membranes offers a sustainable alternative to traditional solvent extraction methods. But in order for biosorption-based REE purification to compete economically, the capacity and specificity of biosorption sites must be enhanced. Although there have been some recent advances in characterizing the genetics of REE-biosorption, the variety and complexity of bacterial membrane surface sites make targeted genetic engineering difficult. Here, we propose using multiple rounds of in vivo random mutagenesis induced by the MP6 plasmid combined with plate-throughput REE-biosorption screening to improve a microbe's capacity and selectivity for biosorbing REE. We engineered a strain of Vibrio natriegens capable of biosorbing 210% more dysprosium compared to the wild-type and produced selectivity improvements of up to 50% between the lightest (lanthanum) and heaviest (lutetium) REE. We believe that mutations we observed in ABC transporters as well as a nonessential protein in the BAM outer membrane β-barrel protein insertion complex likely contribute to some─but almost certainly not all─of the biosorption changes we observed. Given the ease of finding significant biosorption mutants, these results highlight just how many genes likely contribute to biosorption as well as the power of random mutagenesis in identifying genes of interest and optimizing a biological system for a task.

Escherichia coli displays a conserved membrane proteomic response to a range of alcohols

BackgroundAlcohol is a good and environment-friendly fuel that can be microbially produced, capable of eliminating many of the limitations of the present-day fossil fuels. However, the inherent toxic nature of alcohols to the microbial cells leads to end-product inhibition that limits large-scale alcohol production by fermentation. Fundamental knowledge about the stress responses of microorganisms to alcohols would greatly facilitate to improve the microbial alcohol tolerance. The current study elucidates and compares the changes in the membrane proteome of Escherichia coli in response to a range of alcohols.ResultsAlthough alcohol toxicity increased exponentially with alcohol chain length (2–6 carbon), similar stress responses were observed in the inner and outer membrane proteome of E. coli in the presence of 2-, 4- and 6-carbon alcohols at the MIC50. This pertains to: (1) increased levels of inner membrane transporters for uptake of energy-producing metabolites, (2) reduced levels of non-essential proteins, associated with anaerobic, carbon starvation and osmotic stress, for energy conservation, (3) increased levels of murein degrading enzymes (MltA, EmtA, MliC and DigH) promoting cell elongation and 4) reduced levels of most outer membrane β-barrel proteins (LptD, FadL, LamB, TolC and BamA). Major outer membrane β-barrel protein OmpC, which is known to contribute to ethanol tolerance and membrane integrity, was notably reduced by alcohol stress. While LPS is important for OmpC trimerisation, LPS release by EDTA did not lower OmpC levels. This suggests that LPS release, which is reported under alcohol stress, does not contribute to the reduced levels of OmpC in the presence of alcohol.ConclusionsSince alcohol primarily targets the integrity of the membrane, maintenance of outer membrane OmpC levels in the presence of alcohol might help in the survival of E. coli to higher alcohol concentrations. The study provides important information about the membrane protein responses of E. coli to a range of alcohols, which can be used to develop targeted strategies for increased microbial alcohol tolerance and hence bioalcohol production.

Yeast mitochondria can process de novo designed β-barrel proteins.

Mitochondrial outer membrane β-barrel proteins are encoded in the nucleus, translated in the cytosol and then targeted to and imported into the respective organelles. Detailed studies have uncovered the mechanisms involved in the import of these proteins and identified the targeting signals and the cytosolic factors that govern their proper biogenesis. Recently, de novo designed eight-stranded β-barrel proteins (Tmb2.3 and Tmb2.17) were shown to fold and assemble into lipid membranes. To better understand the general aspects of the biogenesis of β-barrel proteins, we investigated the fate of these artificial proteins upon their expression in yeast cells. We demonstrate that although these proteins are de novo designed and are not related to bona fide mitochondrial β-barrel proteins, they were targeted to mitochondria and integrated into the organelle outer membrane. We further studied whether this integration requires components of the yeast mitochondrial import machinery like Tom20, Tom70, Tob55/Sam50 and Mas37/Sam37. Whereas it seems that none of the import receptors was required for the biogenesis of the artificial β-barrel proteins, we observed a strong dependency on the TOB/SAM complex. Collectively, our findings demonstrate that the mitochondrial outer membrane is the preferential location in yeast cells for any membrane-embedded β-barrel protein.

A Specific Guide for Metalloenzyme Designers: Introduction and Evolution of Metal-Coordination Spheres Embedded in Protein Environments.

ConspectusMetalloproteins establish a comprehensive molecular space by combining inorganic cofactors with protein environments. The chemical interplay between a metal element and a protein matrix is remarkable yet elusive, as the chemical properties of metal ions do not directly translate into those of metalloenzymes when placed within a protein matrix. Instead, the biochemical context determines the metal-coordination geometries, reaction kinetics, and thermodynamic parameters, such as redox potential and Lewis acidity/basicity. The molecular design of novel metalloproteins, which involves varying the combination of the two major components, metal ion and protein, allows us to validate our current understanding of their chemical interactions, modulate protein structure and function toward specific directions, and create protein-based functional biomaterials and biocatalysts. Considering theoretically possible protein sequence combinations and several metal elements at several oxidation states, artificial metalloenzyme design is analogous to the drawing of new paintings with multiple colors and brush strokes.Our laboratory has developed minimalistic strategies to transform diverse proteins into artificial metalloproteins with novel architectures or catalytic functions. First, we analyzed the geometric parameters of native Zn-binding sites to introduce a tetrahedral zinc-binding motif to a non-α-helical protein scaffold that shows neither pre-existing affinity to any metal ion nor catalytic activity, such as the β-barrel outer membrane protein OmpF. We created a series of artificial Zn-binding OmpF variants that possess coordinatively unsaturated mononuclear Zn-ligation sites and exhibit hydrolytic activities with esters, β-lactams, and β-glycosides. The OmpF variants were further evolved to zinc-dependent glycosidases, addressing a central question in bioinorganic chemistry: can zinc ions directly associate with the hydrolytic cleavage of glycosidic bonds in sugars?To expand the scope of metalloprotein design, we also utilized a metal-chelating noncanonical amino acid, bipyridine-alanine, and constructed tunable dicopper-dependent oxidases and nickel/iridium-dependent photocatalytic cross-coupling enzymes. These advancements increased the complexity and precision of metalloenzyme design, revealing how protein matrices, in conjunction with metallocofactors in aqueous media, facilitate electron transfer and mediate multiple chemical events, such as proton transfer, substrate access, and product release, while suppressing adventitious reactions.Furthermore, we redesigned the secondary and tertiary metal-coordination spheres of manganese-dependent quercetin dioxygenase and zinc-dependent β-lactamase, respectively. Biochemical characterizations of the evolved enzymes revealed that metal ions harmoniously and selectively cooperate with protein environments such that seemingly unimportant residues play critical roles in tuning the electronic structures of the enzymes or determining the metal-dependent reactivity. Thus, our work establishes the fundamental basis of how metalloenzymes function via long-range intermolecular interactions and how we can control their structure and function in specific directions. Consequently, these novel metalloenzymes have provided possible snapshots of how they could have evolved under controlled chemical environments and potential prospects for how to design more sophisticated and efficient biocatalysts with accuracy at the molecular level.

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

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

Structural basis of BAM-mediated outer membrane β-barrel protein assembly.

The outer membrane structure is common in Gram-negative bacteria, mitochondria and chloroplasts, and contains outer membrane β-barrel proteins (OMPs) that are essential interchange portals of materials1-3. All known OMPs share the antiparallel β-strand topology4, implicating a common evolutionary origin and conserved folding mechanism. Models have been proposed for bacterial β-barrel assembly machinery (BAM) to initiate OMP folding5,6; however, mechanisms by which BAM proceeds to complete OMP assembly remain unclear. Here we report intermediate structures of BAM assembling an OMP substrate, EspP, demonstrating sequential conformational dynamics of BAM during the late stages of OMP assembly, which is further supported by molecular dynamics simulations. Mutagenic in vitro and in vivo assembly assays reveal functional residues of BamA and EspP for barrel hybridization, closure and release. Our work provides novel insights into the common mechanism of OMP assembly.

NlpE Is an OmpA-Associated Outer Membrane Sensor of the Cpx Envelope Stress Response.

Gram-negative bacteria utilize several envelope stress responses (ESRs) to sense and respond to diverse signals within a multilayered cell envelope. The CpxRA ESR responds to multiple stresses that perturb envelope protein homeostasis. Signaling in the Cpx response is regulated by auxiliary factors, such as the outer membrane (OM) lipoprotein NlpE, an activator of the response. NlpE communicates surface adhesion to the Cpx response; however, the mechanism by which NlpE accomplishes this remains unknown. In this study, we report a novel interaction between NlpE and the major OM protein OmpA. Both NlpE and OmpA are required to activate the Cpx response in surface-adhered cells. Furthermore, NlpE senses OmpA overexpression and the NlpE C-terminal domain transduces this signal to the Cpx response, revealing a novel signaling function for this domain. Mutation of OmpA peptidoglycan-binding residues abrogates signaling during OmpA overexpression, suggesting that NlpE signaling from the OM through the cell wall is coordinated via OmpA. Overall, these findings reveal NlpE to be a versatile envelope sensor that takes advantage of its structure, localization, and cooperation with other envelope proteins to initiate adaptation to diverse signals. IMPORTANCE The envelope is not only a barrier that protects bacteria from the environment but also a crucial site for the transduction of signals critical for colonization and pathogenesis. The discovery of novel complexes between NlpE and OmpA contributes to an emerging understanding of the key contribution of OM β-barrel protein and lipoprotein complexes to envelope stress signaling. Overall, our findings provide mechanistic insight into how the Cpx response senses signals relevant to surface adhesion and biofilm growth to facilitate bacterial adaptation.

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.

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.

Structural Model of a Porphyromonas gingivalis type IX Secretion System Shuttle Complex

Porphyromonas gingivalis is a gram-negative oral anaerobic pathogen and is one of the key causative agents of periodontitis. P. gingivalis utilises a range of virulence factors, including the cysteine protease RgpB, to drive pathogenesis and these are exported and attached to the cell surface via the type IX secretion system (T9SS). All cargo proteins possess a conserved C-terminal signal domain (CTD) which is recognised by the T9SS, and the outer membrane β-barrel protein PorV (PG0027/LptO) can interact with cargo proteins as they are exported to the bacterial surface. Using a combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical analyses, machine-learning-based modelling and molecular dynamics (MD) simulations, we present a structural model of a PorV:RgpB-CTD complex from P. gingivalis. This is the first structural insight into CTD recognition by the T9SS and shows how the conserved motifs in the CTD are the primary sites that mediate binding. In PorV, interactions with extracellular surface loops are important for binding the CTD, and together these appear to cradle and lock RgpB-CTD in place. This work provides insight into cargo recognition by PorV but may also have important implications for understanding other aspects of type-IX dependent secretion.

MTCH2 is a mitochondrial outer membrane protein insertase.

In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane β-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction.

Crystal structure of Campylobacter jejuni lipoprotein Cj1090c.

In Gram-negative bacteria, lipopolysaccharide (LPS) is an essential component of the asymmetric outer membrane (OM). LptE is an OM lipoprotein that forms a complex with the β-barrel OM protein, LptD. Incorporation of LPS into the OM outer leaflet is essential for bacterial viability, and mediated by the LptD/E complex. The genome of Campylobacter jejuni, a major foodborne pathogen, contains over 20 putative lipoproteins including Cj1090c. Here, we report the crystal structure of Cj1090c at 2.4 Å resolution, revealing structural evidence for LptE in C. jejuni. The analysis of this crystal structure, along with the genomic context, allows us to propose the C. jejuni LPS transport system for the first time, and permits for discussion of the features of the LptD/E complex of C. jejuni.

The antimicrobial peptide Magainin-2 interacts with BamA impairing folding of E. coli membrane proteins.

Antimicrobial peptides (AMPs) are a unique and diverse group of molecules endowed with a broad spectrum of antibiotics properties that are being considered as new alternative therapeutic agents. Most of these peptides are membrane-active molecules, killing bacteria by membrane disruption. However, recently an increasing number of AMPs was shown to enter bacterial cells and target intracellular processes fundamental for bacterial life. In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent β-barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling, in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. Functional implications of this interactions were investigated by two alternative and complementary approaches. The amount of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation were evaluated by both western blot analysis and quantitative tandem mass spectrometry in Multiple Reaction Monitoring scan mode. In both experiments a gradual decrease in outer membrane proteins production with time was observed as a consequence of Mag-2 treatment. These results suggested BamA as a possible good target for the rational design of new antibiotics since this protein is responsible for a crucial biological event of bacterial life and is absent in humans.