scholarly journals Structure of a Tc holotoxin pore provides insights into the translocation mechanism

2019 ◽  
Vol 116 (46) ◽  
pp. 23083-23090 ◽  
Author(s):  
Daniel Roderer ◽  
Oliver Hofnagel ◽  
Roland Benz ◽  
Stefan Raunser
Keyword(s):  
Pathogenic Bacteria ◽  
Conformational Transition ◽  
Target Cells ◽  
Photorhabdus Luminescens ◽  
C Terminus ◽  
Head Groups ◽  
Toxin Complex ◽  
High Resolution Structure ◽  
Translocation Mechanism ◽  
Resolution Structure

Tc toxins are modular toxin systems of insect and human pathogenic bacteria. They are composed of a 1.4-MDa pentameric membrane translocator (TcA) and a 250-kDa cocoon (TcB and TcC) encapsulating the 30-kDa toxic enzyme (C terminus of TcC). Binding of Tc toxins to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. A high-resolution structure of a holotoxin embedded in membranes is missing, leaving open the question of whether TcB-TcC has an influence on the conformational transition of TcA. Here we show in atomic detail a fully assembled 1.7-MDa Tc holotoxin complex from Photorhabdus luminescens in the membrane. We find that the 5 TcA protomers conformationally adapt to fit around the cocoon during the prepore-to-pore transition. The architecture of the Tc toxin complex allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Importantly, assembly of the holotoxin at the membrane results in spontaneous translocation of the toxic enzyme, indicating that this process is not driven by a proton gradient or other energy source. Mammalian lipids with zwitterionic head groups are preferred over other lipids for the integration of Tc toxins. In a nontoxic Tc toxin variant, we can visualize part of the translocating toxic enzyme, which transiently interacts with alternating negative charges and hydrophobic stretches of the translocation channel, providing insights into the mechanism of action of Tc toxins.

scholarly journals Structure of a Tc holotoxin pore provides insights into the translocation mechanism

2019 ◽  
Author(s):  
Daniel Roderer ◽  
Oliver Hofnagel ◽  
Roland Benz ◽  
Stefan Raunser
Keyword(s):  
Conformational Transition ◽  
Target Cells ◽  
Enzyme Binding ◽  
Ph Shift ◽  
Head Groups ◽  
Toxin Complex ◽  
Translocation Mechanism

AbstractTc toxins are modular toxin systems that are composed of a pentameric membrane translocator (TcA) and a cocoon (TcB and TcC) encapsulating the toxic enzyme. Binding of Tcs to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. Here, we show in atomic detail an assembled Tc toxin complex fromP. luminescensin the membrane. We find that the five TcA protomers conformationally adapt to fit around the cocoon during prepore-to-pore transition. The architecture of the Tc toxin complex also allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Mammalian lipids with zwitterionic head groups are preferred over other lipids for Tc toxin integration. The translocated toxic enzyme, which can be partially visualized, transiently interacts with alternating negative charges and hydrophobic stretches.

scholarly journals Crystallographic study of the 2-thioribothymidine-synthetic complex TtuA–TtuB fromThermus thermophilus

2016 ◽  
Vol 72 (10) ◽  
pp. 777-781 ◽  
Author(s):  
Minghao Chen ◽  
Shun Narai ◽  
Naoki Omura ◽  
Naoki Shigi ◽  
Sarin Chimnaronk ◽  
...  
Keyword(s):  
Thermus Thermophilus ◽  
Covalent Bond ◽  
Transferase Activity ◽  
Structure Model ◽  
Partial Structure ◽  
Data Set ◽  
Crystallographic Study ◽  
C Terminus ◽  
High Resolution Structure ◽  
Resolution Structure

The ubiquitin-like protein TtuB is a sulfur carrier for the biosynthesis of 2-thioribothymidine (s2T) at position 54 in some thermophilic bacterial tRNAs. TtuB captures a S atom at its C-terminus as a thiocarboxylate and transfers it to tRNA by the transferase activity of TtuA. TtuB also functions to suppress s2T formation by forming a covalent bond with TtuA. To explore how TtuB interacts with TtuA and switches between these two different functions, high-resolution structure analysis of the TtuA–TtuB complex is required. In this study, the TtuA–TtuB complex fromThermus thermophiluswas expressed, purified and crystallized. To mimic the thiocarboxylated TtuB, the C-terminal Gly residue was replaced with Cys (G65C) to obtain crystals of the TtuA–TtuB complex. A Zn-MAD data set was collected to a resolution of 2.5 Å. MAD analysis successfully determined eight Zn sites, and a partial structure model composed of four TtuA–TtuB complexes in the asymmetric unit was constructed.

scholarly journals Glycan-dependent two-step cell adhesion mechanism of Tc toxins

2019 ◽  
Author(s):  
Daniel Roderer ◽  
Felix Bröcker ◽  
Oleg Sitsel ◽  
Paulina Kaplonek ◽  
Franziska Leidreiter ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Pathogenic Bacteria ◽  
Host Cells ◽  
Lewis Antigens ◽  
Receptor Binding Domain ◽  
Photorhabdus Luminescens ◽  
Membrane Permeation ◽  
Glycan Array ◽  
Toxin Complex ◽  
Heparan Sulfates

AbstractToxin complex (Tc) toxins are virulence factors widespread in insect and human bacterial pathogens. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and prepore-to-pore transition have been well-described, little is known about receptor binding and cellular uptake of Tcs. Here, we identify two classes of glycans, heparins/heparan sulfates and Lewis antigens, that act as receptors for different TcAs from insect- and human pathogenic bacteria. Glycan array screening and electron cryo microscopy (cryo-EM) structures reveal that all tested TcAs bind unexpectedly with their α-helical part of the shell domain to negatively charged heparins. In addition, TcdA1 from the insect-pathogen Photorhabdus luminescens binds to Lewis antigens with micromolar affinity. A cryo-EM structure of the TcdA1-Lewis X complex reveals that the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a two-step association mechanism of Tc toxins involving glycans on the surface of host cells.

scholarly journals Decoding the Mechanism of Specific RNA Targeting by Ribosomal Methytransferases

2021 ◽  
Author(s):  
Juhi Singh ◽  
Rahul Raina ◽  
Kutti R. Vinothkumar ◽  
Ruchi Anand
Keyword(s):  
Drug Resistance ◽  
Antibiotic Resistance ◽  
Pathogenic Bacteria ◽  
High Fidelity ◽  
Ribosomal Biogenesis ◽  
Structural Elements ◽  
Rna Targeting ◽  
Biochemical Studies ◽  
High Resolution Structure ◽  
Resolution Structure

AbstractMethylation of specific nucleotides is integral for ribosomal biogenesis and serves as a common way to confer antibiotic resistance by pathogenic bacteria. Here, by determining the high-resolution structure of 30S-KsgA by cryo-EM, a state was captured, where KsgA juxtaposes between helices h44 and h45, separating them, thereby enabling remodeling of the surrounded rRNA and allowing the cognate site to enter the methylation pocket. With the structure as a guide, factors that direct the enzyme to its cognate site with high fidelity were unearthed by creating several mutant versions of the ribosomes, where interacting bases in the catalytic helix h45 and surrounding helices h44, h24, and h27 were mutated and evaluated for their methylation efficiency. The biochemical studies delineated specificity hotspots that enable KsgA to achieve an induced fit. This study enables the identification of distal exclusive allosteric pocket and other divergent structural elements in each rMTase, which can be exploited to develop strategies to reverse methylation, mediated drug resistance.

scholarly journals Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile

2021 ◽  
Author(s):  
Akihiro Kawamoto ◽  
Tomohito Yamada ◽  
Toru Yoshida ◽  
Takayuki Kato ◽  
Hideaki Tsuge
Keyword(s):  
High Resolution ◽  
Structural Information ◽  
Host Cells ◽  
Binary Toxin ◽  
Specific Interactions ◽  
Clostridioides Difficile ◽  
Toxin Complex ◽  
High Resolution Structure ◽  
Partial Unfolding ◽  
Resolution Structure

Abstract Besides two large cytotoxins (TcdA and TcdB), certain Clostridioides difficile strains also produce a binary toxin, called C. difficile toxin (CDT) composed of an enzymatic subunit involved in actin ADP-ribosylation (CDTa) and translocation pore (CDTb) that delivers CDTa into host cells through receptor-mediated endocytosis. CDTb is proposed to be a di-heptamer, but its physiological heptameric structure has not been reported to date. Here, we report the CDTa-bound CDTb-pore (heptamer) as a physiological complexes using cryo-EM. The high-resolution structure of the CDTa-bound CDTb-pore at 2.56-Å resolution revealed that CDTa binding to CDTb-pore induces partial unfolding and tilting of the first CDTa a-helix, and the translocation. In the CDTb-pore, the NSS-loop exists in “in” and “out” conformations, suggesting their involvement in substrate translocation through formation of weak, non-specific interactions. This structural information provides insights into drug design against hypervirulent C. difficile strains.

High-resolution structure determination by ALCHEMI

1983 ◽  
Vol 41 ◽  
pp. 178-181 ◽  
Author(s):  
Peter G. Self ◽  
Peter R. Buseck
Keyword(s):  
Site Occupancy ◽  
Real Space ◽  
Unit Cell ◽  
Bragg Angle ◽  
Electron Current ◽  
High Resolution Structure ◽  
Beam Incident ◽  
Crystallographic Planes ◽  
Electron Beam Incident ◽  
Resolution Structure

ALCHEMI (Atom Location by CHanneling Enhanced Microanalysis) enables the site occupancy of atoms in single crystals to be determined. In this article the fundamentals of the method for both EDS and EELS will be discussed. Unlike HRTEM, ALCHEMI does not place stringent resolution requirements on the microscope and, because EDS clearly distinguishes between elements of similar atomic number, it can offer some advantages over HRTEM. It does however, place certain constraints on the crystal. These constraints are: a) the sites of interest must lie on alternate crystallographic planes, b) the projected charge density on the alternate planes must be significantly different, and c) there must be at least one atomic species that lies solely on one of the planes.An electron beam incident on a crystal undergoes elastic scattering; in reciprocal space this is seen as a diffraction pattern and in real space this is a modulation of the electron current across the unit cell. When diffraction is strong (i.e., when the crystal is oriented near to the Bragg angle of a low-order reflection) the electron current at one point in the unit cell will differ significantly from that at another point.

Cathodoluminescence of Catalyst Crystallites

1982 ◽  
Vol 40 ◽  
pp. 664-665
Author(s):  
E.D. Boyes ◽  
P.L. Gai ◽  
D.B. Darby ◽  
C. Warwick
Keyword(s):  
Defect Density ◽  
Chemical Properties ◽  
Operating Conditions ◽  
Semiconductor Materials ◽  
Environmental Cell ◽  
High Resolution Structure ◽  
Commercially Important ◽  
Crystallographic Defects ◽  
Resolution Structure

The extended crystallographic defects introduced into some oxide catalysts under operating conditions may be a consequence and accommodation of the changes produced by the catalytic activity, rather than always being the origin of the reactivity. Operation without such defects has been established for the commercially important tellurium molybdate system. in addition it is clear that the point defect density and the electronic structure can both have a significant influence on the chemical properties and hence on the effectiveness (activity and selectivity) of the material as a catalyst. SEM/probe techniques more commonly applied to semiconductor materials, have been investigated to supplement the information obtained from in-situ environmental cell HVEM, ultra-high resolution structure imaging and more conventional AEM and EPMA chemical microanalysis.

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