RESUMO
The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic ß-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Escherichia coli , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Periplasma/metabolismo , Domínios Proteicos , Dobramento de ProteínaRESUMO
The bacterial flagellum, which facilitates motility, is composed of ~20 structural proteins organized into a long extracellular filament connected to a cytoplasmic rotor-stator complex via a periplasmic rod. Flagellum assembly is regulated by multiple checkpoints that ensure an ordered gene expression pattern coupled to the assembly of the various building blocks. Here, we use epifluorescence, super-resolution, and transmission electron microscopy to show that the absence of a periplasmic protein (FlhE) prevents proper flagellar morphogenesis and results in the formation of periplasmic flagella in Salmonella enterica. The periplasmic flagella disrupt cell wall synthesis, leading to a loss of normal cell morphology resulting in cell lysis. We propose that FlhE functions as a periplasmic chaperone to control assembly of the periplasmic rod, thus preventing formation of periplasmic flagella.
Assuntos
Proteínas de Bactérias , Flagelos , Chaperonas Moleculares , Periplasma , Flagelos/metabolismo , Flagelos/ultraestrutura , Flagelos/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Periplasma/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Salmonella enterica/metabolismo , Salmonella enterica/genética , Microscopia Eletrônica de Transmissão , Proteínas Periplásmicas/metabolismo , Proteínas Periplásmicas/genética , Regulação Bacteriana da Expressão GênicaRESUMO
The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD ß6-ß7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Escherichia coli , Escherichia coli , Periplasma , Fosfolipídeos , Fosfolipídeos/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Periplasma/metabolismo , Transporte Biológico , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Modelos Moleculares , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de MembranaRESUMO
Hydrogen production through water splitting is a vital strategy for renewable and sustainable clean energy. In this study, we developed an approach integrating nanomaterial engineering and synthetic biology to establish a bionanoreactor system for efficient hydrogen production. The periplasmic space (20 to 30 nm) of an electroactive bacterium, Shewanella oneidensis MR-1, was engineered to serve as a bionanoreactor to enhance the interaction between electrons and protons, catalyzed by hydrogenases for hydrogen generation. To optimize electron transfer, we used the microbially reduced graphene oxide (rGO) to coat the electrode, which improved the electron transfer from the electrode to the cells. Native MtrCAB protein complex on S. oneidensis and self-assembled iron sulfide (FeS) nanoparticles acted in tandem to facilitate electron transfer from an electrode to the periplasm. To enhance proton transport, S. oneidensis MR-1 was engineered to express Gloeobacter rhodopsin (GR) and the light-harvesting antenna canthaxanthin. This led to efficient proton pumping when exposed to light, resulting in a 35.6% increase in the rate of hydrogen production. The overexpression of native [FeFe]-hydrogenase further improved the hydrogen production rate by 56.8%. The bionanoreactor engineered in S. oneidensis MR-1 achieved a hydrogen yield of 80.4 µmol/mg protein/day with a Faraday efficiency of 80% at a potential of -0.75 V. This periplasmic bionanoreactor combines the strengths of both nanomaterial and biological components, providing an efficient approach for microbial electrosynthesis.
Assuntos
Grafite , Hidrogênio , Shewanella , Hidrogênio/metabolismo , Shewanella/metabolismo , Shewanella/genética , Grafite/metabolismo , Hidrogenase/metabolismo , Hidrogenase/genética , Transporte de Elétrons , Reatores Biológicos , Biologia Sintética/métodos , Eletrodos , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/genética , Periplasma/metabolismo , Fontes de Energia Bioelétrica/microbiologiaRESUMO
TolC is the outer membrane protein responsible for antibiotic efflux in E. coli. Compared to other outer membrane proteins it has an unusual fold and has been shown to fold independently of commonly used periplasmic chaperones, SurA and Skp. Here we find that the assembly of TolC involves the formation of two folded intermediates using circular dichroism, gel electrophoresis, site-specific disulfide bond formation and radioactive labeling. First the TolC monomer folds, and then TolC assembles into a trimer both in detergent-free buffer and in the presence of detergent micelles. We find that a TolC trimer also forms in the periplasm and is present in the periplasm before it inserts in the outer membrane. The monomeric and trimeric folding intermediates may be used in the future to develop a new approach to antibiotic efflux pump inhibition by targeting the assembly pathway of TolC.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Membrana Transportadoras , Dobramento de Proteína , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Dicroísmo Circular , Periplasma/metabolismo , Multimerização ProteicaRESUMO
BACKGROUND: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation. RESULTS: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L-1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds. CONCLUSION: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.
Assuntos
Dissulfetos , Escherichia coli , Peptídeos , Periplasma , Escherichia coli/metabolismo , Escherichia coli/genética , Periplasma/metabolismo , Dissulfetos/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Aprotinina/metabolismo , Aprotinina/genéticaRESUMO
Technology of production of single-domain antibodies (NANOBODY® molecules, also referred to as nanoantibodies, nAb, or molecules based on other stable protein structures) and their derivatives to solve current problems in biomedicine is becoming increasingly popular. Indeed, the format of one small, highly soluble protein with a stable structure, fully functional in terms of specific recognition, is very convenient as a module for creating multivalent, bi-/oligo-specific genetically engineered targeting molecules and structures. Production of nAb in periplasm of E. coli bacterium is a very convenient and fairly universal way to obtain analytical quantities of nAb for the initial study of the properties of these molecules and selection of the most promising nAb variants. The situation is more complicated with production of bi- and multivalent derivatives of the initially selected nAbs under the same conditions. In this work, extended linker sequences (52 and 86 aa) between the antigen-recognition modules in the cloned expression constructs were developed and applied in order to increase efficiency of production of bispecific nanoantibodies (bsNB) in the periplasm of E. coli bacteria. Three variants of model bsNBs described in this study were produced in the periplasm of bacteria and isolated in soluble form with preservation of functionality of all the protein domains. If earlier our attempts to produce bsNB in the periplasm with traditional linkers no longer than 30 aa were unsuccessful, the extended linkers used here provided a significantly more efficient production of bsNB, comparable in efficiency to the traditional production of original monomeric nAbs. The use of sufficiently long linkers could presumably be useful for increasing efficiency of production of other bsNBs and similar molecules in the periplasm of E. coli bacteria.
Assuntos
Anticorpos Biespecíficos , Escherichia coli , Periplasma , Anticorpos de Domínio Único , Escherichia coli/genética , Escherichia coli/metabolismo , Periplasma/metabolismo , Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/genética , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Antígenos/imunologiaRESUMO
Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 µM) include PBPs (PBP1a, KD = 0.07 µM; PBP5 = 0.4 µM); other lytic transglycosylases (SltB2, KD = 0.09 µM; RlpA, KD = 0.4 µM); a type VI secretion system effector (Tse5, KD = 0.3 µM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 µM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.
Assuntos
Proteínas de Bactérias , Pseudomonas aeruginosa , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Periplasma/metabolismo , Periplasma/enzimologia , Proteínas Periplásmicas/metabolismo , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/química , Glicosiltransferases/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/química , Peptidoglicano/metabolismo , Peptidoglicano/químicaRESUMO
Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.
Assuntos
Agrobacterium tumefaciens , Proteínas de Bactérias , Biofilmes , GMP Cíclico , Pterinas , Biofilmes/crescimento & desenvolvimento , Agrobacterium tumefaciens/metabolismo , Agrobacterium tumefaciens/genética , Pterinas/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteobactérias/metabolismo , Proteobactérias/genética , Cofatores de Molibdênio , Periplasma/metabolismo , Proteínas Periplásmicas/metabolismo , Proteínas Periplásmicas/genética , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas Periplásmicas de Ligação/genética , Regulação Bacteriana da Expressão GênicaRESUMO
Multiheme cytochromes located in different compartments are crucial for extracellular electron transfer in the bacterium Geobacter sulfurreducens to drive important environmental processes and biotechnological applications. Recent studies have unveiled that for particular sets of electron terminal acceptors, discrete respiratory pathways selectively recruit specific cytochromes from both the inner and outer membranes. However, such specificity was not observed for the abundant periplasmic cytochromes, namely the triheme cytochrome family PpcA-E. In this work, the distinctive NMR spectroscopic signatures of these proteins in different redox states were explored to monitor pairwise interactions and electron transfer reactions between each pair of cytochromes. The results showed that the five proteins interact transiently and can exchange electrons between each other revealing intra-promiscuity within the members of this family. This discovery is discussed in the light of the establishment of an effective electron transfer network by this pool of cytochromes. This network is advantageous to the bacteria as it enables the maintenance of the functional working potential redox range within the cells.
Assuntos
Proteínas de Bactérias , Geobacter , Geobacter/metabolismo , Transporte de Elétrons , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citocromos/metabolismo , Citocromos/química , Oxirredução , Periplasma/metabolismo , Periplasma/químicaRESUMO
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
Periplasmic nitrate reductase NapA from Campylobacter jejuni (C. jejuni) contains a molybdenum cofactor (Moco) and a 4Fe-4S cluster and catalyzes the reduction of nitrate to nitrite. The reducing equivalent required for the catalysis is transferred from NapC â NapB â NapA. The electron transfer from NapB to NapA occurs through the 4Fe-4S cluster in NapA. C. jejuni NapA has a conserved lysine (K79) between the Mo-cofactor and the 4Fe-4S cluster. K79 forms H-bonding interactions with the 4Fe-4S cluster and connects the latter with the Moco via an H-bonding network. Thus, it is conceivable that K79 could play an important role in the intramolecular electron transfer and the catalytic activity of NapA. In the present study, we show that the mutation of K79 to Ala leads to an almost complete loss of activity, suggesting its role in catalytic activity. The inhibition of C. jejuni NapA by cyanide, thiocyanate, and azide has also been investigated. The inhibition studies indicate that cyanide inhibits NapA in a non-competitive manner, while thiocyanate and azide inhibit NapA in an uncompetitive manner. Neither inhibition mechanism involves direct binding of the inhibitor to the Mo-center. These results have been discussed in the context of the loss of catalytic activity of NapA K79A variant and a possible anion binding site in NapA has been proposed.
Assuntos
Campylobacter jejuni , Lisina , Nitrato Redutase , Lisina/metabolismo , Lisina/química , Campylobacter jejuni/enzimologia , Campylobacter jejuni/genética , Nitrato Redutase/metabolismo , Nitrato Redutase/química , Nitrato Redutase/genética , Periplasma/metabolismo , Periplasma/enzimologia , BiocatáliseRESUMO
Escherichia coli is often used as a factory to produce recombinant proteins. In many cases, the recombinant protein needs disulfide bonds to fold and function correctly. These proteins are genetically fused to a signal peptide so that they are secreted to the oxidizing environment of the periplasm (where the enzymes required for disulfide bond formation exist). Currently, it is difficult to determine in vivo whether a recombinant protein is efficiently secreted from the cytoplasm and folded in the periplasm or if there is a bottleneck in one of these steps because cellular capacity has been exceeded. To address this problem, we have developed a biosensor that detects cellular stress caused by (1) inefficient secretion of proteins from the cytoplasm and (2) aggregation of proteins in the periplasm. We demonstrate how the fluorescence fingerprint obtained from the biosensor can be used to identify induction conditions that do not exceed the capacity of the cell and therefore do not cause cellular stress. These induction conditions result in more effective biomass and in some cases higher titers of soluble recombinant proteins.
Assuntos
Técnicas Biossensoriais , Escherichia coli , Proteínas Periplásmicas , Técnicas Biossensoriais/métodos , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas Periplásmicas/metabolismo , Proteínas Periplásmicas/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Periplasma/metabolismo , Estresse Fisiológico , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genéticaRESUMO
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
The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with ß-barrel OM proteins (OMPs). Before reaching the OM, precursor unfolded OMPs (uOMPs) must cross the whole cell envelope. A network of periplasmic chaperones and proteases maintains unfolded but folding-competent conformations of these membrane proteins in the aqueous periplasm while simultaneously preventing off-pathway aggregation. These periplasmic proteins utilize different strategies, including conformational heterogeneity, oligomerization, multivalency, and kinetic partitioning, to perform and regulate their functions. Redundant and unique characteristics of the individual periplasmic players synergize to create a protein quality control team capable responding to changing environmental stresses.
Assuntos
Chaperonas Moleculares , Periplasma , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Periplasma/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Dobramento de Proteína , Bactérias Gram-Negativas/metabolismoRESUMO
The Helicobacter pylori Cag type IV secretion system (Cag T4SS) has an important role in the pathogenesis of gastric cancer. The Cag T4SS outer membrane core complex (OMCC) is organized into three regions: a 14-fold symmetric outer membrane cap (OMC) composed of CagY, CagX, CagT, CagM, and Cag3; a 17-fold symmetric periplasmic ring (PR) composed of CagY and CagX; and a stalk with unknown composition. We investigated how CagT, CagM, and a conserved antenna projection (AP) region of CagY contribute to the structural organization of the OMCC. Single-particle cryo-EM analyses showed that complexes purified from ΔcagT or ΔcagM mutants no longer had organized OMCs, but the PRs remained structured. OMCCs purified from a CagY antenna projection mutant (CagY∆AP) were structurally similar to WT OMCCs, except for the absence of the α-helical antenna projection. These results indicate that CagY and CagX are sufficient for maintaining a stable PR, but the organization of the OMC requires CagY, CagX, CagM, and CagT. Our results highlight an unexpected structural independence of two major subdomains of the Cag T4SS OMCC.
Assuntos
Helicobacter pylori , Sistemas de Secreção Tipo IV/química , PeriplasmaRESUMO
The production of disulfide bond-containing recombinant proteins in Escherichia coli has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of E. coli: i) engineered strains with deletions in the disulfide reduction pathways, e.g. SHuffle, and ii) the co-expression of oxidative folding catalysts, e.g. CyDisCo. However, to our knowledge, the relative effectiveness of these strategies has not been properly evaluated. Here, we systematically compare the purified yields of 14 different proteins of interest (POI) that contain disulfide bonds in their native state when expressed in both systems. We also compared the effects of different background strains, commonly used promoters, and two media types: defined and rich autoinduction. In rich autoinduction media, POI which can be produced in a soluble (non-native) state without a system for disulfide bond formation were produced in higher purified yields from SHuffle, whereas all other proteins were produced in higher purified yields using CyDisCo. In chemically defined media, purified yields were at least 10x higher in all cases using CyDisCo. In addition, the quality of the three POI tested was superior when produced using CyDisCo.
Assuntos
Citoplasma , Dissulfetos , Proteínas de Escherichia coli , Escherichia coli , Dobramento de Proteína , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Dissulfetos/metabolismo , Dissulfetos/química , Citoplasma/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Oxirredução , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Periplasma/metabolismo , Periplasma/genética , Meios de Cultura/químicaRESUMO
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).
Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Chaperonas Moleculares/metabolismo , Periplasma/metabolismo , Dobramento de ProteínaRESUMO
Spurred by advances in AI-driven modeling and experimental methods, molecular dynamics simulations are now acting as a platform to integrate these different approaches. This combination of methods is especially useful to understand ß-barrel proteins from the molecular level, e.g., identifying specific interactions with lipids or small molecules, up to assemblies comprised of hundreds of proteins and thousands of lipids. In this minireview, we will discuss recent advances, mainly from the last 5 years, in modeling ß-barrel proteins and their assemblies. These approaches require specific kinds of modeling and potentially different model resolutions that we will first describe in Subheading 1. We will then focus on different aspects of ß-barrel protein modeling: how different types of molecules can diffuse through ß-barrel proteins (Subheading 2); how lipids can interact with these proteins (Subheading 3); how ß-barrel proteins can interact with membrane partners (Subheading 4) or periplasmic extensions and partners (Subheading 5) to form large assemblies.
Assuntos
Proteínas de Membrana , Simulação de Dinâmica Molecular , Periplasma/metabolismo , Lipídeos , Proteínas da Membrana Bacteriana Externa/metabolismoRESUMO
The bacterial flagellum is a complex structure formed by more than 25 different proteins, this appendage comprises three conserved structures: the basal body, the hook and filament. The basal body, embedded in the cell envelope, is the most complex structure and houses the export apparatus and the motor. In situ images of the flagellar motor in different species have revealed a huge diversity of structures that surround the well-conserved periplasmic components of the basal body. The identity of the proteins that form these novel structures in many cases has been elucidated genetically and biochemically, but in others they remain to be identified or characterized. In this work, we report that in the alpha proteobacteria Cereibacter sphaeroides the novel protein MotK along with MotE are essential for flagellar rotation. We show evidence that these periplasmic proteins interact with each other and with MotB2. Moreover, these proteins localize to the flagellated pole and MotK localization is dependent on MotB2 and MotA2. These results together suggest that the role of MotK and MotE is to activate or recruit the flagellar stators to the flagellar structure.