RESUMO
Members of a group of multimeric secretion pores that assemble independently of any known membrane-embedded insertase in Gram-negative bacteria fold into a prepore before membrane-insertion occurs. The mechanisms and the energetics that drive the folding of these proteins are poorly understood. Here, equilibrium unfolding and hydrogen/deuterium exchange monitored by mass spectrometry indicated that a loss of 4-5 kJ/mol/protomer in the N3 domain that is peripheral to the membrane-spanning C domain in the dodecameric secretin PulD, the founding member of this class, prevents pore formation by destabilizing the prepore into a poorly structured dodecamer as visualized by electron microscopy. Formation of native PulD-multimers by mixing protomers that differ in N3 domain stability, suggested that the N3 domain forms a thermodynamic seal onto the prepore. This highlights the role of modest free energy changes in the folding of pre-integration forms of a hyperstable outer membrane complex and reveals a key driving force for assembly independently of the ß-barrel assembly machinery.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Mutação/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Homologia de Sequência de AminoácidosRESUMO
Nanomachines belonging to the type IV filament (Tff) superfamily serve a variety of cellular functions in prokaryotes, including motility, adhesion, electrical conductance, competence and secretion. The type 2 secretion system (T2SS) Tff member assembles a short filament called pseudopilus that promotes the secretion of folded proteins from the periplasm across the outer membrane of Gram-negative bacteria. A combination of structural, biochemical, imaging, computational and in vivo approaches had led to a working model for the assembled nanomachine. High-resolution cryo-electron microscopy and tomography provided the first view of several homologous Tff nanomachines in the cell envelope and revealed the structure of the outer membrane secretin channel, challenging current models of the overall stoichiometry of the T2SS. In addition, recent insights into exoprotein substrate features and interactions with the T2SS have led to new questions about the dynamics of the system and the role of the plasma membrane in substrate presentation. This micro-review will highlight recent advances in the field of type 2 secretion and discuss approaches that can be used to reach a mechanistic understanding of exoprotein recognition, integration into the machine and secretion.
Assuntos
Sistemas de Secreção Tipo II/metabolismo , Sistemas de Secreção Tipo II/ultraestrutura , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Sequência de Bases , Microscopia Crioeletrônica/métodos , Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana/metabolismo , Modelos Moleculares , Periplasma/metabolismo , Ligação Proteica , Dobramento de Proteína , Secretina/química , Relação Estrutura-AtividadeRESUMO
Proteins called secretins form large multimeric complexes that are essential for macromolecular transit across the outer membrane of Gram-negative bacteria. Evidence suggests that the channels formed by some secretin complexes are not tightly closed, but their permeability properties have not been well characterized. Here, we used cell-free synthesis coupled with spontaneous insertion into liposomes to investigate the permeability of the secretin PulD. Leakage assays using preloaded liposomes indicated that PulD allows the efflux of small fluorescent molecules with a permeation cutoff similar to that of general porins. Other secretins were also found to form similar pores. To define the polypeptide region involved in determining the pore size, we analyzed a collection of PulD variants and studied the roles of gates 1 and 2, which were previously reported to affect the pore size of filamentous phage f1 secretin pIV, in assembly and pore formation. Liposome leakage and a novel in vivo assay showed that replacement of the conserved proline residue at position 443 in PulD by leucine increased the apparent size of the pore. The in vitro approach described here could be used to study the pore properties of membrane proteins whose production in vivo is toxic.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Porinas/química , Porinas/metabolismo , Multimerização Proteica , Proteínas da Membrana Bacteriana Externa/genética , Análise Mutacional de DNA , Proteínas de Escherichia coli/genética , Corantes Fluorescentes/metabolismo , Lipossomos/metabolismo , Permeabilidade , Porinas/genéticaRESUMO
Investigations into protein folding are largely dominated by studies on monomeric proteins. However, the transmembrane domain of an important group of membrane proteins is only formed upon multimerization. Here, we use in vitro translation-coupled folding and insertion into artificial liposomes to investigate kinetic steps in the assembly of one such protein, the outer membrane secretin PulD of the bacterial type II secretion system. Analysis of the folding kinetics, measured by the acquisition of distinct determinants of the native state, provides unprecedented evidence for a sequential multistep process initiated by membrane-driven oligomerization. The effects of varying the lipid composition of the liposomes indicate that PulD first forms a "prepore" structure that attains the native state via a conformational switch.
Assuntos
Proteínas da Membrana Bacteriana Externa/biossíntese , Sistemas de Secreção Bacterianos/fisiologia , Klebsiella pneumoniae/metabolismo , Dobramento de Proteína , Multimerização Proteica/fisiologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Klebsiella pneumoniae/química , Klebsiella pneumoniae/genética , Lipossomos/química , Estrutura Quaternária de ProteínaRESUMO
IMPORTANCE: Type IV pili and type II secretion systems are members of the widespread type IV filament (T4F) superfamily of nanomachines that assemble dynamic and versatile surface fibers in archaea and bacteria. The assembly and retraction of T4 filaments with diverse surface properties and functions require the plasma membrane platform proteins of the GspF/PilC superfamily. Generally considered dimeric, platform proteins are thought to function as passive transmitters of the mechanical energy generated by the ATPase motor, to somehow promote insertion of pilin subunits into the nascent pilus fibers. Here, we generate and experimentally validate structural predictions that support the trimeric state of a platform protein PulF from a type II secretion system. The PulF trimers form selective proton or sodium channels which might energize pilus assembly using the membrane potential. The conservation of the channel sequence and structural features implies a common mechanism for all T4F assembly systems. We propose a model of the oligomeric PulF-PulE ATPase complex that provides an essential framework to investigate and understand the pilus assembly mechanism.
Assuntos
Sistemas de Secreção Tipo II , Sistemas de Secreção Tipo II/metabolismo , Klebsiella , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Adenosina Trifosfatases/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismoRESUMO
The mechanosensitive channel MscL of the plasma membrane of bacteria is a homopentamer involved in the protection of cells during osmotic downshock. The MscL protein, a polypeptide of 136 residues, was recently shown to require YidC to be inserted in the inner membrane of E. coli. The insertase YidC is a component of an insertion pathway conserved in bacteria, mitochondria and chloroplasts. MscL insertion was independent of the Sec translocon. Here, we report sucrose gradient centrifugation and freeze-etching microscopy experiments showing that MscL produced in a cell-free system complemented with preformed liposomes is able to insert directly in a pure lipid bilayer. Patch-clamp experiments performed with the resulting proteoliposomes showed that the protein was highly active. In vitro cell-free synthesis targeting to liposomes is a new promising expression system for membrane proteins, including those that might require an insertion machinery in vivo. Our results also question the real role of insertases such as YidC for membrane protein insertion in vivo.
Assuntos
Proteínas de Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Lipídeos/química , Proteínas de Membrana Transportadoras/metabolismo , Bioquímica/métodos , Sistema Livre de Células , Cloroplastos/metabolismo , Escherichia coli/metabolismo , Técnica de Fratura por Congelamento , Bicamadas Lipídicas/química , Lipossomos/química , Mitocôndrias/metabolismo , Osmose , Técnicas de Patch-Clamp , Peptídeos/químicaRESUMO
The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella or Escherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS-LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS-LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Chaperonas Moleculares/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Proteínas Periplásmicas de Ligação/genética , Transporte ProteicoRESUMO
The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.
Assuntos
Evolução Molecular , Mitocôndrias/genética , Mitocôndrias/metabolismo , Sistemas de Secreção Tipo II/genética , Sistemas de Secreção Tipo II/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Eucariotos/classificação , Eucariotos/genética , Eucariotos/metabolismo , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Modelos Moleculares , Naegleria/classificação , Naegleria/genética , Naegleria/metabolismo , Peroxissomos/metabolismo , Filogenia , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Sistemas de Secreção Tipo II/classificaçãoRESUMO
We engineered a class of proteins that binds selected polypeptides with high specificity and affinity. Use of the protein scaffold of Sac7d, belonging to a protein family that binds various ligands, overcomes limitations inherent in the use of antibodies as intracellular inhibitors: it lacks disulfide bridges, is small and stable, and can be produced in large amounts. An in vitro combinatorial/selection approach generated specific, high-affinity (up to 140 pM) binders against bacterial outer membrane secretin PulD. When exported to the Escherichia coli periplasm, they inhibited PulD oligomerization, thereby blocking the type II secretion pathway of which PulD is part. Thus, high-affinity inhibitors of protein function can be derived from Sac7d and can be exported to, and function in, a cell compartment other than that in which they are produced.
Assuntos
Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação a DNA/química , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Modelos Moleculares , Reação em Cadeia da Polimerase , Conformação Proteica , Radioimunoensaio , Ressonância de Plasmônio de SuperfícieRESUMO
Bacterial type 4a pili are dynamic surface filaments that promote bacterial adherence, motility, and macromolecular transport. Their genes are highly conserved among enterobacteria and their expression in enterohemorrhagic Escherichia coli (EHEC) promotes adhesion to intestinal epithelia and pro-inflammatory signaling. To define the molecular basis of EHEC pilus assembly, we determined the structure of the periplasmic domain of its major subunit PpdD (PpdDp), a prototype of an enterobacterial pilin subfamily containing two disulfide bonds. The structure of PpdDp, determined by NMR, was then docked into the density envelope of purified EHEC pili obtained by cryoelectron microscopy (cryo-EM). Cryo-EM reconstruction of EHEC pili at â¼8 Å resolution revealed extremely high pilus flexibility correlating with a large extended region of the pilin stem. Systematic mutagenesis combined with functional and interaction analyses identified charged residues essential for pilus assembly. Structural information on exposed regions and interfaces between EHEC pilins is relevant for vaccine and drug discovery.
Assuntos
Escherichia coli Êntero-Hemorrágica/química , Proteínas de Escherichia coli/química , Proteínas de Fímbrias/química , Fímbrias Bacterianas/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Escherichia coli Êntero-Hemorrágica/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/química , Fímbrias Bacterianas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Simulação de Acoplamento Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática , TermodinâmicaRESUMO
Bacterial type 2 secretion systems (T2SS), type 4 pili, and archaeal flagella assemble fibres from initially membrane-embedded pseudopilin and pilin subunits. Fibre subunits are made as precursors with positively charged N-terminal anchors, whose cleavage via the prepilin peptidase, essential for pilin membrane extraction and assembly, is followed by N-methylation of the mature (pseudo)pilin N terminus. The conserved Glu residue at position 5 (E5) of mature (pseudo)pilins is essential for assembly. Unlike T4 pilins, where E5 residue substitutions also abolish N-methylation, the E5A variant of T2SS pseudopilin PulG remains N-methylated but is affected in interaction with the T2SS component PulM. Here, biochemical and functional analyses showed that the PulM interaction defect only partly accounts for the PulGE5A assembly defect. First, PulGT2A variant, equally defective in PulM interaction, remained partially functional. Furthermore, pseudopilus assembly defect of pulG(E5A) mutant was stronger than that of the pulM deletion mutant. To understand the dominant effect of E5A mutation, we used molecular dynamics simulations of PulGE5A, methylated PulGWT (MePulGWT), and MePulGE5A variant in a model membrane. These simulations pointed to a key role for an intramolecular interaction between the pseudopilin N-terminal amine and E5 to limit polar interactions with membrane phospholipids. N-methylation of the N-terminal amine further limited its interactions with phospholipid head-groups to facilitate pseudopilin membrane escape. By binding to polar residues in the conserved N-terminal region of PulG, we propose that PulM acts as chaperone to promote pseudopilin recruitment and coordinate its membrane extraction with subsequent steps of the fibre assembly process.
Assuntos
Sequência Conservada , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Multimerização Proteica , Sistemas de Secreção Tipo II , Membrana Celular/metabolismo , Simulação de Dinâmica Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosfolipídeos/metabolismo , Ligação ProteicaRESUMO
Like several other large, multimeric bacterial outer membrane proteins (OMPs), the assembly of the Klebsiella oxytoca OMP PulD does not rely on the universally conserved ß-barrel assembly machinery (BAM) that catalyses outer membrane insertion. The only other factor known to interact with PulD prior to or during outer membrane targeting and assembly is the cognate chaperone PulS. Here, in vitro translation-transcription coupled PulD folding demonstrated that PulS does not act during the membrane insertion of PulD, and engineered in vivo site-specific cross-linking between PulD and PulS showed that PulS binding does not prevent membrane insertion. In vitro folding kinetics revealed that PulD is atypical compared to BAM-dependent OMPs by inserting more rapidly into membranes containing E. coli phospholipids than into membranes containing lecithin. PulD folding was fast in diC14:0-phosphatidylethanolamine liposomes but not diC14:0-phosphatidylglycerol liposomes, and in diC18:1-phosphatidylcholine liposomes but not in diC14:1-phosphatidylcholine liposomes. These results suggest that PulD efficiently exploits the membrane composition to complete final steps in insertion and explain how PulD can assemble independently of any protein-assembly machinery. Lipid-assisted assembly in this manner might apply to other large OMPs whose assembly is BAM-independent.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Membrana Celular/química , Proteínas de Escherichia coli/química , Bicamadas Lipídicas/químicaRESUMO
Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system secretin, and PulD, the Klebsiella oxytoca type II secretion system secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Secretina/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Sistemas de Secreção Bacterianos , Microscopia Crioeletrônica , Klebsiella oxytoca/química , Modelos Moleculares , Estrutura Quaternária de Proteína , Pseudomonas aeruginosa/química , Homologia Estrutural de ProteínaRESUMO
The outer membrane portal of the Klebsiella oxytoca type II secretion system, PulD, is a prototype of a family of proteins, the secretins, which are essential components of many bacterial secretion and pilus assembly machines. PulD is a homododecamer with a periplasmic vestibule and an outer chamber on either side of a membrane-spanning region that is poorly resolved by electron microscopy. Membrane insertion involves the formation of a dodecameric membrane-embedded intermediate. Here, we describe an amino acid substitution in PulD that blocks its assembly at this intermediate "prepore" stage. Electron microscopy indicated that the prepore has an apparently normal periplasmic vestibule but a poorly organized outer chamber. A peptide loop around this amino acid appears to be important for the formation/stability of the fully folded complex. A similar assembly intermediate results from creation of the same amino acid substitution in the Pseudomonas aeruginosa secretin XcpQ.
Assuntos
Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Klebsiella oxytoca/química , Proteínas de Membrana/ultraestrutura , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Klebsiella oxytoca/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestrutura , Homologia Estrutural de Proteína , Relação Estrutura-AtividadeRESUMO
The Klebsiella oxytoca lipoprotein PulS might function as either or both a pilot and a docking factor in the outer membrane targeting and assembly of the Type II secretion system secretin PulD. In the piloting model, PulS binds to PulD monomers and targets them to the outer membrane via the lipoprotein sorting pathway components LolA and LolB. In this model, PulS also protects the PulD monomers from proteolysis during transit through the periplasm. In the docking model, PulS is targeted alone to the outer membrane, where it acts as a receptor for PulD monomers, allowing them to accumulate and assemble specifically in this membrane. PulS was shown to dissociate from and/or re-associate freely with PulD multimers in zwitterionic detergent, making it difficult to determine whether PulS remains associated with PulD dodecamers in the outer membrane by co-purification. However, PulD protomers in the dodecamer were shown to be stable in the absence of PulS, indicating that PulS is only required to protect the protease-susceptible monomer. DegP was identified as one of the proteases that could contribute to PulD degradation in the absence of PulS. Studies on the in vitro assembly and targeting of PulD into Escherichia coli membrane vesicles demonstrated its strong preference to insert into the inner membrane, as is the case in vivo in the absence of PulS. However, PulD could be targeted to outer membrane fragments in vitro if they were preloaded with PulS, indicating the technical feasibility of the docking model. We conclude that both modes of action might contribute to efficient outer membrane targeting of PulD in vivo, although the piloting function is likely to predominate.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Klebsiella oxytoca/metabolismo , Chaperonas Moleculares/metabolismo , Inibidores de Proteases/metabolismo , Sistemas de Secreção Bacterianos , Modelos Biológicos , Multimerização Proteica , Transporte Proteico , ProteóliseRESUMO
The C-terminal core domain of the secretin PulD from Klebsiella oxytoca forms heat-resistant dodecameric complexes within less than 10min in an Escherichia coli in vitro transcription-translation system containing liposomes, and is toxic when made in the cytoplasm without a signal peptide. Random mutagenesis of DNA encoding this region of PulD revealed that amino acid changes throughout almost its entire length abolished toxicity. Most of the amino acid substitutions engendered by the mutations retarded or abolished assembly of the dodecameric secretin complex in vitro and/or in the periplasm. Only one of the tested multimerization-defective variants could be rescued by co-production and mixed multimer formation with wild-type secretin in vitro. A three amino acid insertion specifically generated in a region of PulD that was not affected by the spontaneous mutations formed functional multimers that, unlike the wild-type protein, were dissociated by heating in SDS.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Variação Genética , Klebsiella oxytoca/metabolismo , Multimerização Proteica , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Klebsiella oxytoca/química , Klebsiella oxytoca/genética , Mutagênese Insercional , MutaçãoRESUMO
The DNA-binding protein Sac7d was previously modified to bind with high affinity to the N domain of the outer membrane secretin PulD from the bacterium Klebsiella oxytoca. Here, we show that binding of the Sac7d derivatives (affitins) to PulD is sensitive to conformational changes caused by denaturant and by the zwitterionic detergent Zwittergent 3-14 routinely used to extract secretins from outer membranes. This sensitivity to the conformational state of PulD allowed us to use the affitins as probes for the native structure of PulD and to devise protocols for examining in vitro synthesized protein in nonionic detergent and for the affinity purification of native PulD using affitins as ligands. When fused to periplasmic PhoA, three affitins inhibited PulD multimerization in vivo and caused loss of function. In two cases, this was likely to be due to dimerization of the affitin by the bound PhoA, as the effect was absent when the affitins were fused to monomeric MalE. In the third case, the MalE and PhoA moieties probably interfered sterically with PulD protomer interactions and, thereby, inhibited multimerization. None of the affitins tested interacted with PulD at sites of protomer interaction or blocked the secretin channel through which exoproteins cross the outer membrane in the Type II secretion pathway of which PulD is a key component.
Assuntos
Proteínas de Bactérias/química , Klebsiella oxytoca/química , Sondas Moleculares/metabolismo , Secretina/química , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Cromatografia de Afinidade , Dicroísmo Circular , Proteínas de Ligação a DNA/metabolismo , Detergentes/farmacologia , Epitopos/química , Microscopia Eletrônica , Octoxinol/farmacologia , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Secretina/metabolismo , Solubilidade/efeitos dos fármacos , Ureia/farmacologiaRESUMO
Synthesis of the Klebsiella oxytoca outer membrane secretin PulD, or its membrane-associated core domain, in a liposome-supplemented Escherichia coli in vitro transcription-translation system resulted in the formation of multimers that appeared as typical dodecameric secretin rings when examined by negative-stain electron microscopy. Cryo-electron microscopy of unstained liposomes and differential extraction by urea indicated that the secretin particles were inserted into the liposome membranes. When made in the presence of the detergent Brij-35, PulD and the core domain were synthesized as monomers. Both proteins caused almost immediate growth cessation when synthesized in E. coli without a signal peptide. The small amounts of PulD synthesized before cell death appeared as multimers with characteristics similar to those of the normal outer membrane secretin dodecamers. It was concluded that multimerization and membrane insertion are intrinsic properties of secretin PulD that are independent of a specific membrane environment or membrane-associated factors. The closely related Erwinia chrysanthemi secretin OutD behaved similarly to PulD in all assays, but the more distantly related Neisseria meningitidis secretin PilQ did not form multimers either when made in vitro in the presence of liposomes or when made in E. coli without its signal peptide. This is the first report of the apparently spontaneous in vitro assembly and membrane insertion of a large outer membrane protein complex. Spontaneous multimerization and insertion appear to be restricted to outer membrane proteins closely related to PulD.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Klebsiella oxytoca/metabolismo , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Fímbrias/metabolismo , Klebsiella oxytoca/citologia , Klebsiella oxytoca/efeitos dos fármacos , Klebsiella oxytoca/ultraestrutura , Metabolismo dos Lipídeos/efeitos dos fármacos , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Sinais Direcionadores de Proteínas , Estrutura Quaternária de Proteína , Ureia/farmacologiaRESUMO
Previous studies demonstrated that targeting of the dodecameric secretin PulD to the Escherichia coli outer membrane is strictly dependent on the chaperone-like pilotin PulS. Here, we report that PulD multimerization and membrane association in strains producing PulS were unaffected when the levels of the essential outer membrane assembly factor YaeT(Omp85) were reduced by controlled expression of a paraBAD-yaeT transcriptional fusion. This behaviour contrasted markedly to that of the trimeric porin LamB, which remained monomeric under these conditions. Furthermore, resistance to extraction by the detergent Sarkosyl and by urea, and susceptibility to trypsin digestion all suggested that PulD localized to the outer membrane in YaeT-depleted cells. We conclude that, unlike classical beta-barrel outer membrane proteins such as LamB, multimerization of PulD is largely YaeT-independent.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Fusão Gênica Artificial , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Detergentes/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Porinas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Virais/metabolismo , Sarcosina/análogos & derivados , Sarcosina/farmacologia , Tripsina/farmacologia , Ureia/farmacologiaRESUMO
Secretins are a unique class of bacterial multimeric outer membrane proteins that probably differ considerably from other, less complex outer membrane proteins in their overall structure and organization, and in their requirements for outer membrane targeting and assembly factors. In this MicroCommentary, we discuss these differences with respect to the role of a specific class of lipoproteins, often referred to as pilotins, in secretin complex assembly. We compare them with other lipoproteins that play a role in Omp85/YaeT-mediated assembly of more classical outer membrane proteins. One of the examples we have chosen is the Myxococcus Xanthus lipoprotein Tgl. Coculture of cells with and without Tgl allows secretin assembly (and, hence, type IV pilus assembly) in the cells without Tgl, indicating that it can act by cell-to-cell contact or can transfer between cells.