RESUMO
Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a ß-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 ß-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.
Assuntos
Proteínas da Membrana Bacteriana Externa , Chaperonas Moleculares , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/química , Transporte Proteico , Dobramento de Proteína , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Negativas/genética , Membrana Externa Bacteriana/metabolismo , Modelos Moleculares , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Canais de Translocação SEC/metabolismo , Canais de Translocação SEC/genética , Canais de Translocação SEC/química , Periplasma/metabolismoRESUMO
Transmembrane ß barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the ß 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 ß barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "ß signal" motif of EspP to correctly orient ß strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated ß 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 ß sheets progressively fold toward BamA to form a ß barrel. Along with in vivo experiments that tracked ß barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate ß barrel folding.
Assuntos
Proteínas da Membrana Bacteriana Externa/ultraestrutura , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/metabolismo , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
The ß-barrel assembly machine (BAM) integrates ß-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. An essential BAM subunit (BamA) catalyzes integration by promoting the formation of a hybrid-barrel intermediate state between its own ß-barrel domain and that of its client proteins. Here we show that in addition to catalyzing the integration of ß-barrel proteins, BamA functions as a polypeptide export channel. In vivo structural mapping via intermolecular disulfide crosslinking showed that the extracellular "passenger" domain of a member of the "autotransporter" superfamily of virulence factors traverses the OM through the BamA ß-barrel lumen. Furthermore, we demonstrate that a highly conserved residue within autotransporter ß-barrels is required to position the passenger inside BamA to initiate translocation and that during translocation, the passenger stabilizes the hybrid-barrel state. Our results not only establish a new function for BamA but also unify the divergent functions of BamA and other "Omp85" superfamily transporters.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico , Sequência Conservada , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , TriptofanoRESUMO
The Omp85 protein superfamily is found in the outer membrane (OM) of all gram-negative bacteria and eukaryotic organelles of bacterial origin. Members of the family catalyze both the membrane insertion of ß-barrel proteins and the translocation of proteins across the OM. Although the mechanism(s) by which these proteins function is unclear, striking new insights have emerged from recent biochemical and structural studies. In this review we discuss the entire Omp85 superfamily but focus on the function of the best-studied member, BamA, which is an essential and highly conserved component of the bacterial barrel assembly machinery (BAM). Because BamA has multiple functions that overlap with those of other Omp85 proteins, it is likely the prototypical member of the Omp85 superfamily. Furthermore, BamA has become a protein of great interest because of the recent discovery of small-molecule inhibitors that potentially represent an important new class of antibiotics.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Escherichia coli , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Peptídeos/metabolismo , Dobramento de ProteínaRESUMO
Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique ß 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 ß 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 ß strand 4 in the N-terminal fragment and ß 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 ß barrel). In contrast, we observed only weak disulfide bonding between ß strand 1 in the N-terminal fragment and ß strand 8 in the C-terminal fragment that would form a closed or circularly permutated ß barrel. Our results not only demonstrate that ß barrels begin to fold into a ß-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.
Assuntos
Proteínas da Membrana Bacteriana Externa , Escherichia coli , Proteínas da Membrana Bacteriana Externa/síntese química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Conformação Proteica em Folha beta , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
Bacteroides fragilis toxin (BFT) is a protein secreted by enterotoxigenic (ETBF) strains of B. fragilis. BFT is synthesized as a proprotein (proBFT) that is predicted to be a lipoprotein and that is cleaved into two discrete fragments by a clostripain-like protease called fragipain (Fpn). In this study, we obtained evidence that Fpn cleaves proBFT following its transport across the outer membrane. Remarkably, we also found that the disruption of the fpn gene led to a strong reduction in the level of >100 other proteins, many of which are predicted to be lipoproteins, in the culture medium of an ETBF strain. Experiments performed with purified Fpn provided direct evidence that the protease releases at least some of these proteins from the cell surface. The observation that wild-type cells outcompeted an fpn- strain in co-cultivation assays also supported the notion that Fpn plays an important role in cell physiology and is not simply dedicated to toxin biogenesis. Finally, we found that purified Fpn altered the adhesive properties of HT29 intestinal epithelial cells. Our results suggest that Fpn is a broad-spectrum protease that not only catalyzes the protein secretion on a wide scale but that also potentially cleaves host cell proteins during colonization.
Assuntos
Toxinas Bacterianas/metabolismo , Bacteroides fragilis/metabolismo , Metaloendopeptidases/metabolismo , Peptídeo Hidrolases/metabolismo , Bacteroides fragilis/genética , Cisteína Endopeptidases/metabolismo , Lipoproteínas/metabolismo , Peptídeo Hidrolases/fisiologiaRESUMO
OsmY is a widely conserved but poorly understood 20 kDa periplasmic protein. Using a folding biosensor, we previously obtained evidence that OsmY has molecular chaperone activity. To discover natural OsmY substrates, we screened for proteins that are destabilized and thus present at lower steady-state levels in an osmY-null strain. The abundance of an outer membrane protein called antigen 43 was substantially decreased and its ß-barrel domain was undetectable in the outer membrane of an osmY-null strain. Antigen 43 is a member of the diffuse adherence family of autotransporters. Like strains that are defective in antigen 43 production, osmY-null mutants failed to undergo cellular autoaggregation. In vitro, OsmY assisted in the refolding of the antigen 43 ß-barrel domain and protected it from added protease. Finally, an osmY-null strain that expressed two members of the diffuse adherence family of autotransporters that are distantly related to antigen 43, EhaA and TibA, contained reduced levels of the proteins and failed to undergo cellular autoaggregation. Taken together, our results indicate that OsmY is involved in the biogenesis of a major subset of autotransporters, a group of proteins that play key roles in bacterial pathogenesis.
Assuntos
Adesinas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Dobramento de Proteína , Sistemas de Secreção Tipo V/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Chaperonas Moleculares/metabolismo , Proteínas Periplásmicas de Ligação/genética , Domínios Proteicos/fisiologiaRESUMO
Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "ß-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively but often requires non-physiological conditions and time scales and is strongly influenced by properties of the lipid bilayer, including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the ß-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel-phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA, and another model protein (OmpG) folded at significantly different rates and that an α-helix embedded inside the EspP ß-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Bicamadas Lipídicas/química , Proteínas Ligadas a Lipídeos/química , Proteínas Ligadas a Lipídeos/genética , Lipossomos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Porinas/química , Porinas/genética , Porinas/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
Although the barrel assembly machinery (Bam) complex has been shown to facilitate the insertion of ß barrel proteins into the bacterial outer membrane (OM), the stage at which ß barrels fold is unknown. Here, we describe insights into ß barrel assembly that emerged from an analysis of a member of the autotransporter family of OM proteins (EspP) in Escherichia coli. EspP contains an extracellular 'passenger' domain that is translocated across the OM and then released from the covalently linked ß barrel domain in an intra-barrel cleavage reaction. We found that the mutation of an unusual lipid-exposed lysine residue impairs a previously unidentified late folding step that follows both the membrane insertion of the ß barrel domain and the secretion of the passenger domain but that precedes proteolytic maturation. Our results demonstrate that ß barrel assembly can be completed at a post-insertion stage and raise the possibility that interactions with membrane lipids can promote folding in vivo. Furthermore, by showing that the passenger domain is secreted before the ß barrel domain is fully assembled, our results also provide evidence against the long-standing hypothesis that autotransporters are autonomous protein secretion systems.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Serina Endopeptidases/metabolismo , Sistemas de Secreção Tipo V/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Lisina/genética , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Domínios Proteicos/genética , Dobramento de Proteína , Análise de Sequência de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/genética , Sistemas de Secreção Tipo V/química , Sistemas de Secreção Tipo V/genéticaRESUMO
Almost all bacterial outer membrane proteins (OMPs) contain a ß barrel domain that serves as a membrane anchor, but the assembly and quality control of these proteins are poorly understood. Here, we show that the introduction of a single lipid-facing arginine residue near the middle of the ß barrel of the Escherichia coli OMPs OmpLA and EspP creates an energy barrier that impedes membrane insertion. Although several unintegrated OmpLA mutants remained insertion-competent, they were slowly degraded by the periplasmic protease DegP. Two EspP mutants were also gradually degraded by DegP but were toxic because they first bound to the Bam complex, an essential heteroligomer that catalyzes the membrane insertion of OMPs. Interestingly, another EspP mutant likewise formed a prolonged, deleterious interaction with the Bam complex but was protected from degradation and eventually inserted into the membrane in a native conformation. The different types of interactions between the EspP mutants and the Bam complex that we observed may correspond to distinct stages in OMP assembly. Our results show that sequences that significantly delay assembly are disfavored not only because unintegrated OMPs are subjected to degradation, but also because OMPs that assemble slowly can form dominant-negative interactions with the Bam complex.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Fosfolipases A1/genética , Serina Endopeptidases/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Periplasma/metabolismo , Proteínas Periplásmicas/metabolismo , Fosfolipases A1/metabolismo , Domínios Proteicos/genética , Dobramento de Proteína , Estrutura Terciária de Proteína/genética , Serina Endopeptidases/metabolismoRESUMO
The recognition of a C-terminal motif in E. coli SecM ((150)FXXXXWIXXXXGIRAGP(166)) inside the ribosome tunnel causes translation arrest, but the mechanism of recognition is unknown. Whereas single mutations in this motif impair recognition, we demonstrate that new arrest-inducing peptides can be created through remodeling of the SecM C terminus. We found that R163 is indispensable but that flanking residues that vary in number and position play an important secondary role in translation arrest. The observation that individual SecM variants showed a distinct pattern of crosslinking to ribosomal proteins suggests that each peptide adopts a unique conformation inside the tunnel. Based on the results, we propose that translation arrest occurs when the peptide conformation specified by flanking residues moves R163 into a precise intratunnel location. Our data indicate that translation arrest results from extensive communication between SecM and the tunnel and help to explain the striking diversity of arrest-inducing peptides found throughout nature.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/genética , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Fatores de Transcrição/química , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Mapeamento de Interação de Proteínas , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Autotransporters are a large superfamily of cell surface proteins produced by Gram-negative bacteria that consist of an N-terminal extracellular domain ('passenger domain') and a C-terminal ß-barrel domain that resides in the outer membrane (OM). Although it was originally proposed that the passenger domain is translocated across the OM through a channel formed exclusively by the covalently linked ß-barrel domain, this idea has been strongly challenged by a variety of observations. Recent experimental results have suggested a new model in which both the translocation of the passenger domain and the membrane integration of the ß-barrel domain are facilitated by the Bam complex, a highly conserved heteroligomer that plays a general role in OM protein assembly. Other factors, including periplasmic chaperones and inner membrane proteins, have also recently been implicated in the biogenesis of at least some members of the autotransporter superfamily. New results have raised intriguing questions about the energetics of the secretion reaction and the relationship between the assembly of autotransporters and the assembly of other classes of OM proteins. Concomitantly, new mechanistic and structural insights have expanded the utility of the autotransporter pathway for the surface display of heterologous peptides and proteins of interest.
Assuntos
Sistemas de Translocação de Proteínas/metabolismo , Sistemas de Secreção Tipo V/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Modelos Moleculares , Dobramento de Proteína , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Autotransporter (AT) proteins provide a diverse array of important virulence functions to Gram-negative bacterial pathogens, and have also been adapted for protein surface display applications. The 'autotransporter' moniker refers to early models that depicted these proteins facilitating their own translocation across the bacterial outer membrane. Although translocation is less autonomous than originally proposed, AT protein segments upstream of the C-terminal transmembrane ß-barrel have nevertheless consistently been found to contribute to efficient translocation and/or folding of the N-terminal virulence region (the 'passenger'). However, defining the precise secretion functions of these AT regions has been complicated by the use of multiple overlapping and ambiguous terms to define AT sequence, structural, and functional features, including 'autochaperone', 'linker' and 'junction'. Moreover, the precise definitions and boundaries of these features vary among ATs and even among research groups, leading to an overall murky picture of the contributions of specific features to translocation. Here we propose a unified, unambiguous nomenclature for AT structural, functional and conserved sequence features, based on explicit criteria. Applied to 16 well-studied AT proteins, this nomenclature reveals new commonalities for translocation but also highlights that the autochaperone function is less closely associated with a conserved sequence element than previously believed.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Bactérias Gram-Negativas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Biológico , Sequência Conservada , Bactérias Gram-Negativas/química , Chaperonas Moleculares/metabolismo , Fatores de VirulênciaRESUMO
Autotransporters are a large class of virulence proteins produced by Gram-negative bacteria. They contain an N-terminal extracellular ("passenger") domain that folds into a ß-helical structure and a C-terminal ß-barrel ("ß") domain that anchors the protein to the outer membrane. Because the periplasm lacks ATP, the source of energy that drives passenger domain secretion is unknown. The prevailing model postulates that vectorial folding of the ß-helix in the extracellular space facilitates unidirectional secretion of the passenger domain. In this study we used a chimeric protein composed of the 675-residue receptor-binding domain (RD) of the Bordetella pertussis adenylate cyclase toxin CyaA fused to the C terminus of the Escherichia coli O157:H7 autotransporter EspP to test this hypothesis. The RD is a highly acidic, repetitive polypeptide that is intrinsically disordered in the absence of calcium. Surprisingly, we found that the RD moiety was efficiently secreted when it remained in an unfolded conformation. Furthermore, we found that neutralizing or reversing the charge of acidic amino acid clusters stalled translocation in the vicinity of the altered residues. These results challenge the vectorial folding model and, together with the finding that naturally occurring passenger domains are predominantly acidic, provide evidence that a net negative charge plays a significant role in driving the translocation reaction.
Assuntos
Toxina Adenilato Ciclase/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Bordetella pertussis/enzimologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Conformação Proteica , Dobramento de Proteína , Serina Endopeptidases/metabolismo , Bordetella pertussis/genética , Biologia Computacional , Escherichia coli O157 , Cinética , Proteínas de Membrana Transportadoras/química , Oligonucleotídeos/genética , Plasmídeos/genéticaRESUMO
Autotransporters are bacterial virulence factors that contain an N-terminal extracellular ("passenger") domain and a C-terminal ß barrel ("ß") domain that anchors the protein to the outer membrane. The ß domain is required for passenger domain secretion, but its exact role in autotransporter biogenesis is unclear. Here we describe insights into the function of the ß domain that emerged from an analysis of mutations in the Escherichia coli O157:H7 autotransporter EspP. We found that the G1066A and G1081D mutations slightly distort the structure of the ß domain and delay the initiation of passenger domain translocation. Site-specific photocrosslinking experiments revealed that the mutations slow the insertion of the ß domain into the outer membrane, but do not delay the binding of the ß domain to the factor that mediates the insertion reaction (the Bam complex). Our results demonstrate that the ß domain does not simply target the passenger domain to the outer membrane, but promotes translocation when it reaches a specific stage of assembly. Furthermore, our results provide evidence that the Bam complex catalyzes the membrane integration of ß barrel proteins in a multistep process that can be perturbed by minor structural defects in client proteins.
Assuntos
Escherichia coli O157/fisiologia , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/fisiologia , Serina Endopeptidases/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/fisiologia , Escherichia coli O157/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Genes Bacterianos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/genética , Fatores de Virulência/genética , Fatores de Virulência/fisiologiaRESUMO
Autotransporters are a superfamily of virulence proteins produced by Gram-negative bacteria. They consist of an N-terminal ß-helical domain ("passenger domain") that is secreted into the extracellular space and a C-terminal ß-barrel domain ("ß-domain") that anchors the protein to the outer membrane. Because the periplasm lacks ATP, vectorial folding of the passenger domain in a C-to-N-terminal direction has been proposed to drive the secretion reaction. Consistent with this hypothesis, mutations that disrupt the folding of the C terminus of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP have been shown to cause strong secretion defects. Here, we show that point mutations introduced at specific locations near the middle or N terminus of the EspP ß-helix that perturb folding also impair secretion, but to a lesser degree. Surprisingly, we found that even multiple mutations that potentially abolish the stability of several consecutive rungs of the ß-helix only moderately reduce secretion efficiency. Although these results provide evidence that the free energy derived from passenger domain folding contributes to secretion efficiency, they also suggest that a significant fraction of the energy required for secretion is derived from another source.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Substituição de Aminoácidos , Membrana Celular/metabolismo , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Serina Endopeptidases/genéticaRESUMO
Autotransporters are bacterial virulence factors that consist of an N-terminal extracellular ("passenger") domain and a C-terminal ß barrel domain ("ß domain") that resides in the outer membrane. Here we used an in vivo site-specific photocrosslinking approach to gain insight into the mechanism by which the ß domain is integrated into the outer membrane and the relationship between ß domain assembly and passenger domain secretion. We found that periplasmic chaperones and specific components of the ß barrel assembly machinery (Bam) complex interact with the ß domain of the Escherichia coli O157:H7 autotransporter extracellular serine protease P (EspP) in a temporally and spatially regulated fashion. Although the chaperone Skp initially interacted with the entire ß domain, BamA, BamB, and BamD subsequently interacted with discrete ß domain regions. BamB and BamD remained bound to the ß domain longer than BamA and therefore appeared to function at a later stage of assembly. Interestingly, we obtained evidence that the completion of ß domain assembly is regulated by an intrinsic checkpoint mechanism that requires the completion of passenger domain secretion. In addition to leading to a detailed model of autotransporter biogenesis, our results suggest that the lipoprotein components of the Bam complex play a direct role in the membrane integration of ß barrel proteins.
Assuntos
Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Periplasma/metabolismo , Serina Endopeptidases/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação/genética , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli O157/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Imunoprecipitação , Lipoproteínas/metabolismo , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Mutação , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Ligação Proteica/efeitos da radiação , Estrutura Terciária de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/genética , Fatores de Tempo , Raios UltravioletaRESUMO
The translocation and assembly module (TAM) has been proposed to play a crucial role in the assembly of a small subset of outer membrane proteins (OMPs) in Proteobacteria based on experiments conducted in vivo using tamA and tamB mutant strains and in vitro using biophysical methods. TAM consists of an OMP (TamA) and a periplasmic protein that is anchored to the inner membrane by a single α helix (TamB). Here we examine the function of the purified E. coli complex in vitro after reconstituting it into proteoliposomes. We find that TAM catalyzes the assembly of four model OMPs nearly as well as the ß-barrel assembly machine (BAM), a universal heterooligomer that contains a TamA homolog (BamA) and that catalyzes the assembly of almost all E. coli OMPs. Consistent with previous results, both TamA and TamB are required for significant TAM activity. Our study provides direct evidence that TAM can function as an independent OMP insertase and describes a new method to gain insights into TAM function.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Proteolipídeos/metabolismo , Transporte ProteicoRESUMO
Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded ß-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal ß-barrel domain. Challenging the current dogma, we find that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring ß-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.
Assuntos
Proteínas da Membrana Bacteriana Externa , Multimerização Proteica , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Periplasma/metabolismo , Domínios Proteicos , Membrana Externa Bacteriana/metabolismo , Modelos Moleculares , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genéticaRESUMO
Mutations in the ribosomal protein L22 that impair peptide-mediated translation arrest in Escherichia coli have been shown to reduce the expression of several genes, including secA, which encodes an ATPase that drives protein export via the Sec pathway. Here, we used a comparative proteomic approach to obtain insight into the global effects of the L22(Δ82-84) mutation on gene expression and protein synthesis. While the mutation did not affect or modestly affected the level of most soluble proteins, it dramatically reduced the level of antigen 43 (Ag43), a secreted virulence factor that promotes autoaggregation. The reduced protein concentration correlated with a sharp decrease in the abundance and stability of Ag43 mRNA. We found that the overexpression of secA or the inactivation of genes that encode presecretory and membrane proteins restored Ag43 production in the L22 mutant strain. Furthermore, impairment of the Sec pathway in a wild-type strain reduced Ag43 production but did not significantly affect the synthesis of other presecretory proteins. Taken together, these results indicate that Ag43 gene expression is exquisitely sensitive to the status of the Sec machinery and strongly suggest that the L22 mutation decreases the Ag43 concentration indirectly by reducing secA expression. Our results imply the existence of a novel regulatory mechanism in which the efficiency of protein export is coupled to gene expression and help to explain the modulation of SecA synthesis that has been observed in response to secretion stress.