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Nature Communications Aug 2021Lipoproteins are important for bacterial growth and antibiotic resistance. These proteins use lipid acyl chains attached to the N-terminal cysteine residue to anchor on...
Lipoproteins are important for bacterial growth and antibiotic resistance. These proteins use lipid acyl chains attached to the N-terminal cysteine residue to anchor on the outer surface of cytoplasmic membrane. In Gram-negative bacteria, many lipoproteins are transported to the outer membrane (OM), a process dependent on the ATP-binding cassette (ABC) transporter LolCDE which extracts the OM-targeted lipoproteins from the cytoplasmic membrane. Lipid-anchored proteins pose a unique challenge for transport machinery as they have both hydrophobic lipid moieties and soluble protein component, and the underlying mechanism is poorly understood. Here we determined the cryo-EM structures of nanodisc-embedded LolCDE in the nucleotide-free and nucleotide-bound states at 3.8-Å and 3.5-Å resolution, respectively. The structural analyses, together with biochemical and mutagenesis studies, uncover how LolCDE recognizes its substrate by interacting with the lipid and N-terminal peptide moieties of the lipoprotein, and identify the amide-linked acyl chain as the key element for LolCDE interaction. Upon nucleotide binding, the transmembrane helices and the periplasmic domains of LolCDE undergo large-scale, asymmetric movements, resulting in extrusion of the captured lipoprotein. Comparison of LolCDE and MacB reveals the conserved mechanism of type VII ABC transporters and emphasizes the unique properties of LolCDE as a molecule extruder of triacylated lipoproteins.
Topics: ATP-Binding Cassette Transporters; Acylation; Adenosine Triphosphate; Bacterial Outer Membrane Proteins; Binding Sites; Cell Membrane; Cryoelectron Microscopy; Escherichia coli; Hydrophobic and Hydrophilic Interactions; Lipoproteins; Mutation; Periplasm; Protein Conformation; Protein Transport
PubMed: 34344901
DOI: 10.1038/s41467-021-24965-1 -
Journal of Molecular Biology Jul 2021Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein...
Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MBP) from Escherichia coli. We used single-molecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.
Topics: Binding Sites; Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Ligands; Models, Molecular; Mutation; Periplasmic Binding Proteins; Protein Binding; Protein Conformation; Protein Stability; Single Molecule Imaging
PubMed: 33957147
DOI: 10.1016/j.jmb.2021.167036 -
Journal of Bacteriology Sep 2019OmpA-like proteins are involved in the stabilization of the outer membrane, resistance to osmotic stress, and pathogenesis. In , OmpA2 forms a physiologically relevant...
OmpA-like proteins are involved in the stabilization of the outer membrane, resistance to osmotic stress, and pathogenesis. In , OmpA2 forms a physiologically relevant concentration gradient that forms by an uncharacterized mechanism, in which the gradient orientation depends on the position of the gene locus. This suggests that OmpA2 is synthesized and translocated to the periplasm close to the position of the gene and that the gradient forms by diffusion of the protein from this point. To further understand how the OmpA2 gradient is established, we determined the localization and mobility of the full protein and of its two structural domains. We show that OmpA2 does not diffuse and that both domains are required for gradient formation. The C-terminal domain binds tightly to the cell wall and the immobility of the full protein depends on the binding of this domain to the peptidoglycan; in contrast, the N-terminal membrane β-barrel diffuses slowly. Our results support a model in which once OmpA2 is translocated to the periplasm, the N-terminal membrane β-barrel is required for an initial fast restriction of diffusion until the position of the protein is stabilized by the binding of the C-terminal domain to the cell wall. The implications of these results on outer membrane protein diffusion and organization are discussed. Protein concentration gradients play a relevant role in the organization of the bacterial cell. The protein OmpA2 forms an outer membrane polar concentration gradient. To understand the molecular mechanism that determines the formation of this gradient, we characterized the mobility and localization of the full protein and of its two structural domains an integral outer membrane β-barrel and a periplasmic peptidoglycan binding domain. Each domain has a different role in the formation of the OmpA2 gradient, which occurs in two steps. We also show that the OmpA2 outer membrane β-barrel can diffuse, which is in contrast to what has been reported previously for several integral outer membrane proteins in , suggesting a different organization of the outer membrane proteins.
Topics: Bacterial Outer Membrane; Bacterial Outer Membrane Proteins; Caulobacter crescentus; Diffusion; Gene Expression Regulation, Bacterial; Protein Folding
PubMed: 31209077
DOI: 10.1128/JB.00177-19 -
MBio Oct 2022In Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM)...
In Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM) polysaccharide export (OPX) type translocon exports the polysaccharide across the OM. The paradigm OPX protein Wza of Escherichia coli is an octamer in which the eight C-terminal domains form an α-helical OM pore and the eight copies of the three N-terminal domains (D1 to D3) form a periplasmic cavity. In synthase-dependent pathways, the OM translocon is a 16- to 18-stranded β-barrel protein. In Myxococcus xanthus, the secreted polysaccharide EPS (exopolysaccharide) is synthesized in a Wzx/Wzy-dependent pathway. Here, using experiments, phylogenomics, and computational structural biology, we identify and characterize EpsX as an OM 18-stranded β-barrel protein important for EPS synthesis and identify AlgE, a β-barrel translocon of a synthase-dependent pathway, as its closest structural homolog. We also find that EpsY, the OPX protein of the EPS pathway, consists only of the periplasmic D1 and D2 domains and completely lacks the domain for spanning the OM (herein termed a OPX protein). , EpsX and EpsY mutually stabilize each other and interact in pulldown experiments supporting their direct interaction. Based on these observations, we propose that EpsY and EpsX make up and represent a third type of translocon for polysaccharide export across the OM. Specifically, in this composite translocon, EpsX functions as the OM-spanning β-barrel translocon together with the periplasmic OPX protein EpsY. Based on computational genomics, similar composite systems are widespread in Gram-negative bacteria. Bacteria secrete a wide variety of polysaccharides that have critical functions in, e.g., fitness, surface colonization, and biofilm formation and in beneficial and pathogenic human-, animal-, and plant-microbe interactions. In Gram-negative bacteria, export of these chemically diverse polysaccharides across the outer membrane depends on two known translocons, i.e., an outer membrane OPX protein in Wzx/Wzy- and ABC transporter-dependent pathways and an outer membrane 16- to 18-stranded β-barrel protein in synthase-dependent pathways. Here, using a combination of experiments in Myxococcus xanthus, phylogenomics, and computational structural biology, we provide evidence supporting that a third type of translocon can export polysaccharides across the outer membrane. Specifically, in this translocon, an outer membrane-spanning β-barrel protein functions together with an entirely periplasmic OPX protein that completely lacks the domain for spanning the OM. Computational genomics support that similar composite systems are widespread in Gram-negative bacteria.
Topics: ATP-Binding Cassette Transporters; Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Gram-Negative Bacteria; Periplasm; Polysaccharides, Bacterial
PubMed: 35972145
DOI: 10.1128/mbio.02032-22 -
Nature Communications Jul 2021Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan...
Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.
Topics: Amino Acid Motifs; Bacterial Capsules; Catalytic Domain; Cryoelectron Microscopy; Cytoplasm; Escherichia coli; Escherichia coli Proteins; Mass Spectrometry; Membrane Proteins; Models, Molecular; Periplasm; Phosphorylation; Polysaccharides, Bacterial; Protein Conformation, alpha-Helical; Protein-Tyrosine Kinases; Tyrosine
PubMed: 34272394
DOI: 10.1038/s41467-021-24652-1 -
Nature Communications Oct 2021The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has...
The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has limited the application of continuous evolution technologies to antibodies and other disulfide-containing proteins. Here we describe periplasmic phage-assisted continuous evolution (pPACE), a system for continuous evolution of protein-protein interactions in the disulfide-compatible environment of the E. coli periplasm. We first apply pPACE to rapidly evolve novel noncovalent and covalent interactions between subunits of homodimeric YibK protein and to correct a binding-defective mutant of the anti-GCN4 Ω-graft antibody. We develop an intein-mediated system to select for soluble periplasmic expression in pPACE, leading to an eight-fold increase in soluble expression of the Ω-graft antibody. Finally, we evolve disulfide-containing trastuzumab antibody variants with improved binding to a Her2-like peptide and improved soluble expression. Together, these results demonstrate that pPACE can rapidly optimize proteins containing disulfide bonds, broadening the applicability of continuous evolution.
Topics: Binding Sites; Cloning, Molecular; Coliphages; Directed Molecular Evolution; Disulfides; Escherichia coli; Escherichia coli Proteins; Gene Expression; Genetic Vectors; Inteins; Methyltransferases; Models, Molecular; Periplasm; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Disulfide-Isomerases; Protein Interaction Domains and Motifs; Protein Splicing; Receptor, ErbB-2; Recombinant Proteins; Trastuzumab
PubMed: 34645844
DOI: 10.1038/s41467-021-26279-8 -
Life Science Alliance Jun 2024The 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...
The 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 Δ or Δ mutants no longer had organized OMCs, but the PRs remained structured. OMCCs purified from a CagY antenna projection mutant (CagYAP) 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.
Topics: Helicobacter pylori; Type IV Secretion Systems; Periplasm
PubMed: 38631913
DOI: 10.26508/lsa.202302560 -
PloS One 2022The symport of lactose and H+ is an important physiological process in E. coli, for it is closely related to cellular energy supply. In this paper, we review, extend and...
The symport of lactose and H+ is an important physiological process in E. coli, for it is closely related to cellular energy supply. In this paper, we review, extend and analyse a newly proposed cotransport model that takes the "leakage" phenomenon (uncoupled particle translocation) into account and also satisfies the static head equilibrium condition. Then, we use the model to study the equilibrium properties, including equilibrium solution and the time required to reach equilibrium, of the symport process of E. coli LacY protein, when varying the parameters of the initial state of cotransport system. It can be found that in our extended model, H+ and lactose will reach their equilibrium state separately, and when "leakage" exists, it linearly affects the equilibrium solution, which is a useful property that the original model does not have. We later investigated the effect of the volume of periplasm and cytoplasm on the equilibrium properties. For a certain E. coli cell, as it continues to lose water and contract, the time for cytoplasm pH to be stabilized by symport increases monotonically when the cell survives. Finally, we reproduce the experimental data from a literature to verify the validity of the extension in this symport process. The above phenomena and other findings in this paper may help us to not only further validate or improve the model, but also deepen our understanding of the cotransport process of E. coli LacY protein.
Topics: Biological Transport; Cytoplasm; Escherichia coli; Escherichia coli Proteins; Hydrogen-Ion Concentration; Ion Transport; Kinetics; Lactose; Membrane Transport Proteins; Monosaccharide Transport Proteins; Periplasm; Protons; Reproducibility of Results; Symporters; Thermodynamics
PubMed: 35120164
DOI: 10.1371/journal.pone.0263286 -
Proceedings of the National Academy of... May 2022The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron...
The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron tomography, we report that the P. gingivalis T9SS consists of 18 PorM dimers arranged as a large, caged ring in the periplasm. Near the outer membrane, PorM dimers interact with a PorKN ring complex of ∼52 nm in diameter. PorMKN translocation complexes of a given T9SS adopt distinct conformations energized by the proton motive force, suggestive of different activation states. At the inner membrane, PorM associates with a cytoplasmic complex that exhibits 12-fold symmetry and requires both PorM and PorL for assembly. Activated motors deliver substrates across the outer membrane via one of eight Sov translocons arranged in a ring. The T9SSs are unique among known secretion systems in bacteria and eukaryotes in their assembly as supramolecular machines composed of apparently independently functioning translocation motors and export pores.
Topics: Bacterial Proteins; Bacterial Secretion Systems; Periplasm; Porphyromonas gingivalis; Virulence Factors
PubMed: 35471908
DOI: 10.1073/pnas.2119907119 -
Frontiers in Microbiology 2022and species oxidize methanol pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca-dependent MDH (MxaF) and...
and species oxidize methanol pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca-dependent MDH (MxaF) and lanthanide (Ln)-dependent MDH (XoxF), whose expression is regulated by the availability of Ln. A set of a siderophore, TonB-dependent receptor, and an ABC transporter that resembles the machinery for iron uptake is involved in the solubilization and transport of Ln. The transport of Ln into the cytosol enhances XoxF expression. A unique protein named lanmodulin from strain AM1 was identified as a specific Ln-binding protein, and its biological function was implicated to be an Ln shuttle in the periplasm. In contrast, it remains unclear how Ln levels in the cells are maintained, because Ln is potentially deleterious to cellular systems due to its strong affinity to phosphate ions. In this study, we investigated the function of a lanmodulin homolog in strain 22A. The expression of a gene encoding lanmodulin () was induced in response to the presence of La. A recombinant LanM underwent conformational change upon La binding. Phenotypic analyses on deletion mutant and overexpressing strains showed that LanM is not necessary for the wild-type and XoxF-dependent mutant's methylotrophic growth. We found that expression was regulated by MxcQE (a two-component regulator for MxaF) and TonB_Ln (a TonB-dependent receptor for Ln). The expression level of was altered to be negatively dependent on Ln concentration in ∆ whereas it was constant in the wild type. Furthermore, when exposed to La, ∆ showed an aggregating phenotype, cell membrane impairment, La deposition in the periplasm evidenced by electron microscopy, differential expression of proteins involved in membrane integrity and phosphate starvation, and possibly lower La content in the membrane vesicle (MV) fractions. Taken together, we concluded that lanmodulin is involved in the complex regulation mechanism of MDHs and homeostasis of cellular Ln levels by facilitating transport and MV-mediated excretion.
PubMed: 35814700
DOI: 10.3389/fmicb.2022.921636