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Journal of Bacteriology Jun 2019About one-third of the proteins encoded by the bacterial genomes that have been sequenced to date are proteins of "unknown function." Studies aimed at defining the...
About one-third of the proteins encoded by the bacterial genomes that have been sequenced to date are proteins of "unknown function." Studies aimed at defining the biological functions of these proteins represent an important frontier in prokaryotic biology. The study presented by J. Herrou et al. (J Bacteriol 201:e00134-19, 2019) in this issue of the provides an excellent example of how to pursue such studies and define a new virulence determinant for an important zoonotic pathogen.
Topics: Brucella; Periplasm; Periplasmic Proteins; Virulence; Virulence Factors
PubMed: 30936369
DOI: 10.1128/JB.00216-19 -
EcoSal Plus Feb 2019The formation of disulfide bonds is critical to the folding of many extracytoplasmic proteins in all domains of life. With the discovery in the early 1990s that... (Review)
Review
The formation of disulfide bonds is critical to the folding of many extracytoplasmic proteins in all domains of life. With the discovery in the early 1990s that disulfide bond formation is catalyzed by enzymes, the field of oxidative folding of proteins was born. played a central role as a model organism for the elucidation of the disulfide bond-forming machinery. Since then, many of the enzymatic players and their mechanisms of forming, breaking, and shuffling disulfide bonds have become understood in greater detail. This article summarizes the discoveries of the past 3 decades, focusing on disulfide bond formation in the periplasm of the model prokaryotic host .
Topics: Catalysis; Disulfides; Escherichia coli; Escherichia coli Proteins; Oxidation-Reduction; Periplasm; Periplasmic Proteins; Protein Disulfide-Isomerases; Protein Folding
PubMed: 30761987
DOI: 10.1128/ecosalplus.ESP-0012-2018 -
Proceedings of the National Academy of... Dec 2018The lactose permease of (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward...
The lactose permease of (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.
Topics: Binding Sites; Crystallography, X-Ray; Cytoplasm; Escherichia coli; Escherichia coli Proteins; Galactosides; Membrane Transport Proteins; Monosaccharide Transport Proteins; Periplasm; Symporters
PubMed: 30478058
DOI: 10.1073/pnas.1816267115 -
BioMed Research International 2019All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable... (Review)
Review
All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an "off" to an "on" state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.
Topics: Biosensing Techniques; Periplasm; Periplasmic Binding Proteins; Protein Domains; Protein Engineering
PubMed: 30719443
DOI: 10.1155/2019/4798793 -
MBio Jun 2021We demonstrate here that the acquisition of DNase resistance by transforming DNA, often assumed to indicate transport to the cytoplasm, reflects uptake to the periplasm,...
We demonstrate here that the acquisition of DNase resistance by transforming DNA, often assumed to indicate transport to the cytoplasm, reflects uptake to the periplasm, requiring a reevaluation of conclusions about the roles of several proteins in transformation. The new evidence suggests that the transformation pilus is needed for DNA binding to the cell surface near the cell poles and for the initiation of uptake. The cellular distribution of the membrane-anchored ComEA of Bacillus subtilis does not dramatically change during DNA uptake as does the unanchored ComEA of and . Instead, our evidence suggests that ComEA stabilizes the attachment of transforming DNA at localized regions in the periplasm and then mediates uptake, probably by a Brownian ratchet mechanism. Following that, the DNA is transferred to periplasmic portions of the channel protein ComEC, which plays a previously unsuspected role in uptake to the periplasm. We show that the transformation endonuclease NucA also facilitates uptake to the periplasm and that the previously demonstrated role of ComFA in the acquisition of DNase resistance derives from the instability of ComGA when ComFA is deleted. These results prompt a new understanding of the early stages of DNA uptake for transformation. Transformation is a widely distributed mechanism of bacterial horizontal gene transfer that plays a role in the spread of antibiotic resistance and virulence genes and more generally in evolution. Although transformation was discovered nearly a century ago and most, if not all the proteins required have been identified in several bacterial species, much remains poorly understood about the molecular mechanism of DNA uptake. This study uses epifluorescence microscopy to investigate the passage of labeled DNA into the compartment between the cell wall and the cell membrane of Bacillus subtilis, a necessary early step in transformation. The roles of individual proteins in this process are identified, and their modes of action are clarified.
Topics: Bacillus subtilis; Biological Transport; Cell Membrane; DNA, Bacterial; Membrane Proteins; Periplasm; Transformation, Bacterial
PubMed: 34126763
DOI: 10.1128/mBio.01061-21 -
Journal of Structural Biology Sep 2022The CusS histidine kinase is a member of Escherichia coli two-component signal transduction system, engaged in a response to copper ions excess in the cell periplasm....
The CusS histidine kinase is a member of Escherichia coli two-component signal transduction system, engaged in a response to copper ions excess in the cell periplasm. The periplasmic sensor domain of CusS binds the free copper ions and the CusS kinase core phosphorylates the cognate CusR which regulates transcription of the efflux pomp CusCBA. A small amount of copper ions is indispensable for the aerobic cell metabolism. Nonetheless, its excess in the cytoplasm generates damaging and reactive hydroxyl radicals. For that reason, understanding the bacterial copper sensing mechanisms can contribute to reducing bacterial copper-resistance and developing bactericidal copper-based materials. The crystal structure of the CusS kinase core was solved at the resolution of 1.4 Å. The cytoplasmic catalytic core domains formed a homodimer. Based on the obtained structure, the intramolecular and intermolecular interactions crucial for the mechanism of CusS autophosphorylation were described.
Topics: Copper; Escherichia coli; Escherichia coli Proteins; Histidine Kinase; Periplasm
PubMed: 35907487
DOI: 10.1016/j.jsb.2022.107883 -
Cell Jul 2016It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the...
It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client's affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins.
Topics: Carrier Proteins; Entropy; Escherichia coli; Escherichia coli Proteins; Hydrophobic and Hydrophilic Interactions; Molecular Chaperones; Periplasm; Periplasmic Proteins; Protein Folding; Static Electricity
PubMed: 27293188
DOI: 10.1016/j.cell.2016.05.054 -
Scientific Reports Sep 2018The sugar transporter Lactose permease (LacY) of Escherichia coli has become a prototype to understand the underlying molecular details of membrane transport. Crystal...
The sugar transporter Lactose permease (LacY) of Escherichia coli has become a prototype to understand the underlying molecular details of membrane transport. Crystal structures have trapped the protein in sugar-bound states facing the periplasm, but with narrow openings unable to accommodate sugar. Therefore, the molecular details of sugar uptake remain elusive. In this work, we have used extended simulations and metadynamics sampling to explore a putative sugar-uptake pathway and associated free energy landscape. We found an entrance at helix-pair 2 and 11, which involved lipid head groups and residues Gln 241 and Gln 359. Furthermore, the protein displayed high flexibility on the periplasmic side of Phe 27, which is located at the narrowest section of the pathway. Interactions to Phe 27 enabled passage into the binding site, which was associated with a 24 ± 4 kJ/mol binding free energy in excellent agreement with an independent binding free energy calculation and experimental data. Two free energy minima corresponding to the two possible binding poses of the lactose analog β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG) were aligned with the crystal structure-binding pocket. This work outlines the chemical environment of a putative periplasmic sugar pathway and paves way for understanding substrate affinity and specificity in LacY.
Topics: Cell Membrane; Lipid Bilayers; Membrane Transport Proteins; Molecular Dynamics Simulation; Periplasm; Protein Conformation; Protein Transport; Thermodynamics
PubMed: 30254312
DOI: 10.1038/s41598-018-32624-7 -
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