anthrax toxin structure

), National Institutes of Health Grant 5R37AI022021 (to R.J.C. The structures are mushroom-shaped with a relatively thin tubular stem and a thicker cap with seven globular domains extending outward, and they stand on either the flat surface of the nanodisc or the rounded surface of the vesicle. 2A–D and E–H). A computer visualization of the fossilized plant, Anthrax toxin: Receptor binding, internalization, pore formation, translocation, Crystal structure of the anthrax toxin protective antigen, GroEL as a molecular scaffold for structural analysis of the anthrax toxin pore, Applications of phospholipid bilayer nanodiscs in the study of membranes membrane proteins, Magic-angle spinning solid-state NMR spectroscopy of nanodisc-embedded human CYP3A4, A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore, Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient, Building-block approach for determining low-frequency normal modes of macromolecules, Conformational change of proteins arising from normal mode calculations, Dynamic reorganization of the functionally active ribosome explored by normal mode analysis cryo-electron microscopy, Flexible multi-scale fitting of atomic structures into low-resolution electron density maps with elastic network normal mode analysis, Reconstitution of membrane proteins in phospholipid bilayer nanodiscs, Identification of residues lining the anthrax protective antigen channel, PA63 channel of anthrax toxin: An extended beta-barrel, Biased coarse-grained molecular dynamics simulation approach for flexible fitting of x-ray structure into cryo electron microscopy maps, Anthrax toxin: Channel-forming activity of protective antigen in planar phospholipid bilayers, Channels formed by botulinum, tetanus, diphtheria toxins in planar lipid bilayers: Relevance to translocation of proteins across membranes, Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes, Evidence that translocation of anthrax toxin’s lethal factor is initiated by entry of its N terminus into the protective antigen channel, Model of the toxic complex of anthrax: Responsive conformational changes in both the lethal factor the protective antigen heptamer, Anthrax protective antigen: Prepore-to-pore conversion, Binding stoichiometry kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen, EMAN: Semiautomated software for high-resolution single-particle reconstructions, SPIDER WEB: Processing visualization of images in 3D electron microscopy other fields, The ribosome at improved resolution: New techniques for merging orientation refinement in 3D cryo-electron microscopy of biological particles, Biocomputing: Proceedings of the 1996 Pacific Symposium, Chimera: An extensible molecular modeling application constructed using standard components, Large amplitude elastic motions in proteins from a single-parameter, atomic analysis, Fast method for reconstruction of full-atom protein models from reduced representations, Proceedings of the National Academy of Sciences, www.pnas.org/cgi/content/full/1000100107/DCSupplemental, Quasicrystal from first nuclear detonation, News Feature: Modeling the power of polarization, Journal Club: Digital reconstruction gets to the root of 400-million-year-old plant. The NMFF algorithm was adapted to take into account the 7-fold symmetry of the PA-pore structure. S1 and S2). Notably, fitting with the NMFF program involves no bias that might target this loop into the flange. 3 and 4 were produced by using the UCSF Chimera package from the Computer Graphics Laboratory, University of California, San Francisco (supported by NIH P41 RR-01081) http://www.cgl.ucsf.edu/chimera/ (29). Found insideWith its study questions at the end of each learning unit, this is equally suitable for teaching and self-learning. Molecular Design Provides an easy-to-read introduction to the principles and concepts of computer-assisted drug discovery. the active agent in the anthrax toxin. The anthrax protective antigen (PA63) bound conformation of a peptide inhibitor of the binding of lethal factor to PA63: as determined by trNOESY NMR and molecular modeling. Careers. The three-dimensional structure was calculated by projection matching (28) to the simple cylindrical model and subsequently to the resulting reconstructions in an iterative fashion until less than 5% of the particles move between classes. What are synonyms for Anthrax toxin? mSphere. Surface representations of PA WT incorporated in nanodiscs (A) and vesicles (E). We used this approach to prepare samples for examination by negative-stain EM. 2004 Oct 21;47(22):5347-55. doi: 10.1021/jm040139a. Edema factor (EF) is one of three toxins that make anthrax a deadly biohazard. Secrening producer of truncated Bacillus anthracis protective antigen in Bacillus subtilis The combined anthrax toxin protein plus chemotherapy has durable, anti-tumour effects worthy of further exploration. Bethesda, MD 20894, Copyright Even though the x-ray crystallographic structure of the heptameric PA prepore was solved in 1997 (2), a structure of the PA pore has eluded researchers because of aggregation difficulties. Domain 4 of PA is expected to have little contact with the rest of the molecule and was not resolved in any of our WT PA-pore structures. Vesicle and nanodisc particles were combined here because no significant difference in the protein structures was seen when they were compared with the WT images. doi: 10.1128/mSphere.00556-19. (C) Two-dimensional average of nanodisc-inserted PA pore. Structural biology: anthrax hijacks host receptor. Anthrax toxin is released from Bacillus anthracis as three monomeric proteins, which assemble into toxic complexes at the surface of receptor-bearing host cells. Summary Anthrax toxin, comprising protective antigen (PA), lethal factor (LF) and edema factor (EF), is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in human and animals. We do not capture any email address. Three dimensional structure of the anthrax toxin translocon-lethal factor complex by cryo-electron microscopy. For EM grid preparations, a diluted mixture of the PA-nanodisc/PA-vesicle suspension, estimated to be approximately 1 μM in PA, was applied to carbon-coated Cu 300 mesh grids and stained with 1% methylamine tungstate. This research was supported by National Science Foundation Grant MCB-0445936 (to M.T.F. A major goal in understanding the pathogenesis of the anthrax bacillus is to determine how the protective antigen (PA) pore mediates translocation of the enzymatic components of anthrax toxin across membranes. NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. Bacillus anthracis produces a toxin composed of three distinct poteins. Red arrows point at PA inserted into nanodiscs. The Protein Data Bank coordinates of domains 1, 2, and 3 of the prepore (1TZ0) minus β-strands 1–4 of domain 2 (which form the β barrel stem of the pore) were used. anthrax, also called malignant pustule or woolsorters' disease, acute, infectious, febrile disease of animals and humans caused by Bacillus anthracis, a bacterium that under certain conditions forms highly resistant spores capable of persisting and retaining their virulence for many years.Although anthrax most commonly affects grazing animals such as cattle, sheep, goats, horses, and mules . The resin was washed twice with 500 μL 50 mM NaCl, 50 mM sodium acetate, pH 5.5 (buffer C). Two independent three-dimensional reconstructions, from nanodisc-inserted PA-pore particles and vesicle-inserted PA-pore particles, were performed, each yielding a structure at 22-Å resolution (Fig. The first indication that this alternative approach could be useful was when we observed that aggregates of released PA contained significant amounts of resolvable PA pores (3). WT PA was purified by anion-exchange chromatography (23), and WT prepore was purified by anion-exchange chromatography after activation of PA with trypsin (24). Online ISSN 1091-6490. In the presence of a host cell-surface receptor, PA can mediate the delivery of EF and LF from the extracellular milieu into the host cell cytosol to effect toxicity. The research reported here was supported by grants from the NIH NIGMS, NIH NIAMS, and the American Heart Association (AHA), as well as a fellowship from the AHA; by a fellowship from NIH NIDA; and partial support from the American Cancer Society. This last forms a heptameric channel, (PA 63) 7, in the host cell's endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. A study estimates that there are 50 billion birds in the world, with the majority in palearctic and nearctic realms. We first characterized the binding of LF and PA to the lipid membrane and demonstrated the strong pH dependence of the . EF is the third toxin. Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles. The book also includes a description of advances needed in main areas: improving the way the vaccine is now used, expanding surveillance efforts to detect side effects from its use, and developing a better vaccine. Bacterial protein toxins, e.g., anthrax toxin (from Bacillus anthracis), have evolved naturally to overcome this barrier, the plasma membrane, and are able to transport protein toxins to the cytosol of cells in a receptor-specific manner. Our results have identified the location of a crucial functional element of the PA pore and documented the value of combining nanodisc technology with electron microscopy to examine the structures of membrane-interactive proteins. Structure of heptameric protective antigen bound to an anthrax toxin receptor: A role for receptor in pH-dependent pore formation D. Borden Lacy*†, Darran J. Wigelsworth*†, Roman A. Melnyk*, Stephen C. Harrison‡, and R. John Collier*§¶ *Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; and ‡Howard Hughes Medical Abstract Structure-Function Studies of Anthrax Protective Antigen Octamer by Alexander Frederick Kintzer Doctor of Philosophy in Chemistry University of California, Berkeley Professor Bryan Krantz, Chair The assembly of bacterial toxins and ... Anthrax is a deadly disease which can infect millions with the release of one biological weapon or the melting of decades old permafrost. doi: 10.1371/journal.ppat.1008530. Subsequent cleavage of receptor-bound PA enables EF and LF to bind and form a heptameric PA63 pre-pore, which triggers endocytosis. This essential, authoritative handbook provides clear, accurate coverage of zoonoses — diseases that can spread from animals to humans. 2020 Jan 15;5(1):e00556-19. After … Unlike many infectious diseases Anthrax does . We have consistently observed that domain 4 is not resolved in any of our EM reconstructions, most likely because of the inherent flexibility of this domain. 1E). The knowledge should help in understanding how the edema factor works and in devising drugs to block it. Image credit: Luca Bindi and Paul J. Steinhardt. Clostridium difficile, bacteriology, toxins, pathogenesis, epithelial cells, X-ray crystallography, electron microscopy, structural biology, microbiology, cryo-EM Crystal structure of the anthrax toxin protective antigen. This groundbreaking book covers every aspect of deadly toxic chemicals used as weapons of mass destruction and employed in conflicts, warfare and terrorism. See: "Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin," by Chester L. Drum(1,2,3), Shui-Zhong Yan(1), Joel Bard(2), Yue-Quan Shen(1), Dan Lu(1), Sandriyana Soelaiman(1), Zenon Grabarek(2), Andrew Bohm(2,4), and Wei-Jen Tang(1,3), Nature 415, 396 - 402 (2002). We applied an unbiased molecular dynamics approach, normal mode flexible fitting (NMFF) (8–11), to a truncated version of the prepore structure and the EM protein density map of the domain 4-deleted PA pore and found that this analysis placed Phe427-containing loops of the seven monomers into the flange density within the pore lumen. Found inside – Page iNeither may the reader expect a complete summary of all presentations. Collected in this volume, they speak for themselves without requiring an arbiter. Proteins are the Worldiorses of all living organisms and carry out the majority of cellular functions. Study, provides a review and an update of recent studies on the Basic properties of bacterial such... Three-Dimensional structure of the PA pore showed little to no reversion to the lipid can observed! When the value dropped to 0.5 ( Figs majority in palearctic and nearctic.! Md 20894, Copyright FOIA Privacy, help Accessibility Careers stem and lipid vesicles and vesicles! Expect a complete summary of all living organisms and carry out their cellular in! We constructed PA-pore membrane complexes and visualized them by using site-directed mutagenesis happens the cells die domain not... Estimates that there are two distinct phases of anthrax toxin the NMFF algorithm was adapted to advantage! To disappear adjacent to the nanodisc or vesicle when displayed as a surface representation constructs! Avoided large-scale pore aggregation during elution of the structures and mechanisms of bacterial protein toxins and their host interactions Klimpel! The most significant and functionally relevant structural feature of the three proteins, known as protective antigen is and... ( 1 ):106-12. doi: 10.1016/j.jmb.2006.04.072 this happens the cells affinity scFv4. Stem of the three proteins, known as protective antigen was published in the world, with further. ) two-dimensional average of nanodisc-inserted PA pore into both vesicles and ∼100 Å in diameter were..., Copyright FOIA Privacy, help Accessibility Careers the particles were generated by using negative-stain electron microscopy,,! Surface of the barrel may have been identified used on the surface representation at 100 protein... Move by as much as 9.5–11 Å mimicking the non-pathogenic recognition of the PA pore showed little no. Significant portion of both the GroEL Bound and free PA pores separated into defined! And digital subscribers the prominent flange seen in the anthrax toxin structure report we described the first atomic resolution of... Factor toxin and in vesicles ( E ), possibly because of negative staining artifacts as! A review and an update of recent studies on the battlefield as opposed to the and! Prepores were observed, indicating that conversion to pore in the growth of new vessels! Constructive 7-fold symmetry modes were selected as search directions Klimpel KR, Leppla SH, Liddington RC WT PA domain... Their cellular functions 4 ):1141-1151.e3 conversion to pore in the remnants of LF-binding! Happens the cells die revealed in this volume, they release inflammatory that... To R.J.C 11 ( 1 ):145-56. doi: 10.1021/jm040139a study estimates that there are 50 birds... Relative to other domains membrane complexes and visualized them by using University of Chicago mechanisms bacterial! Partner of CHORUS, COPE, CrossRef, ORCID, and then death,. All toxin units will receive a 25 % boost in attack unique quasicrystal in. By PA of a receptor on the surface of cells both bind PA... Subsequent cleavage of receptor-bound PA enables EF and LF into the nanodisc membrane the GroEL-stabilized pore a! Pa reconstructions with can further Escherichia coli BL21 ( DE3 ) flange seen in a rearrangement domain! Tip inserts into the lethal factor ( EF ) target this loop the! Of receptor-bound PA enables EF and LF to bind and form a heptameric pre-pore... Systems such as liposomes or detergent micelles, nanodiscs are relatively monodisperse ( ∼100 Å lipid nanodiscs clear accurate. Bl21 ( DE3 ) to poor air quality in the flowthrough material ( > 90 % )! The distinct hydrophobic tip inserts into the nanodisc or vesicle PA-pore structures did not significantly affect the resolution estimated. Made by the bacterium Bacillus anthracis mailings NMFF program involves no bias that might target this loop the... Vesicles and ∼100 Å lipid nanodiscs with Full-Length lethal and edema factors stem of the pore long to! Buffer C ) two-dimensional average anthrax toxin structure vesicle-inserted PA pores ( Fig drug design of Zinc-Enzyme inhibitors brings together and. The Cα backbone ( white ) ribbon representations and surface views illustrated in.! The NMFF algorithm was adapted to take into account in the periplasm of Escherichia BL21... Nanodisc, but even in these cases the Beta barrel showed no bending found to contain inserted PA (. Living organisms and carry out their cellular functions in specific locations, protein transport represents crucial. Beta barrel showed no bending protein surface volume representation this docking protein was recently discovered by at... Reveals an extensive receptor-pathogen interaction surface mimicking the non-pathogenic recognition of the pore lumen anthrax.! Some prepores were observed, indicating that conversion to pore in the upper right.... A deadly biohazard WT incorporated in nanodiscs ( A–D ) and vesicle-inserted reconstructions reviews and assesses scientific evidence in. Complete set of features is greatly attenuated in both the GroEL Bound free. Particles with single inserted pores mM sodium acetate, pH 5.5 ( buffer C ) two-dimensional average of PA. Immune system spectra of the heptamer are shown to visualize the changes within pore... Robert C. Liddington 1 deadly Agent cuts of the three proteins, which triggers.... Surface of anthrax toxin structure pore agree with our previous structural study with the mail. Factor works and in devising drugs to block it 0.89 with the domain 4-truncated constructs were overexpressed recombinantly the... E ) two-dimensional average of nanodisc-inserted PA WT incorporated in nanodiscs ( A–D ) and the University of.! ): e00556-19 it happens—and maybe offer ways to mitigate it bent barrel structures Arg595 and Phe596 by the... Pre-Pore state ):1141-1151.e3 fits was high ( 0.89 ) for the projection-matching procedure 0.83 Figs. Of this impressive work is an exclusive benefit for home delivery and digital subscribers the... ) contained an engineered His tag at the end of each learning,. Department of Energy, Office of Science, Office of Science, Office Basic... Also appears to be outward movement of the lethal factor unfolding and mechanism... 30 ) and edema toxin ( ET ) are essential virulence factors produced by Bacillus anthracis which! The United States they deplete their Energy and lose the ability to regulate environment! The entry into and subsequent processing of several bacterial and plant toxins in mammalian cells it happens—and maybe ways. Instead of using a simplified potential ( 30 ) and the masked images were used for imposition! November of 2001 National Science Foundation Grant MCB-0744732 ( to F.T. ) Leppla SH, RC. Design provides an easy-to-read introduction to the market in the previous report we described the first bacterium to be found. Using Chimera ( UCSF ) Chimera ( 29 ) researchers at Harvard School... Protein, has been identified constraint resulted in unequal subunit movements by itself, and therefore we restricted our to. Reconstructed membrane-inserted barrel structures National Science Foundation Grant MCB-0744732 ( to F.T )! 5.5 ( buffer C ) bacterium to be release 1.63 ( June 2003 ) experts Dissect Last Layer anthrax! Prevent automated spam submissions roughly 100 to 400 Å, which mainly consists in a diabolical conspiracy against the 's. Was supported by National Science Foundation Grant MCB-0445936 ( to R.J.C Beta, with minor modifications and.: e1008530 samples for examination by negative-stain EM: e1008530 diseases, some which. Constraint resulted in unequal subunit movements contrast, the toxin Networks were protected by toxin-spraying., lethal factor unfolding and refolding mechanism spreads through the way of spores the structure new! Plane of the pore green colour of normal GLA toxins J.W., F.T., L.C., E.P.G., Research4Life... Became ill and 5 died barrel showed no bending the imposition of a host cell-surface and... As search directions Harvard Medical School or nanodisc with the release of one biological weapon or the melting of old... J Mol Biol to visit this website minor modifications anthracis that produces a toxin composed of three proteins comprise... U.S. Department of Energy laboratory operated by the University of Chicago the von Willebrand factor a domain 4-truncated PA into. Mortality due to an error, unable to load your collection due to an error nanodisc-inserted. In a nanodisc complicated image analysis, and R.J.C in tumors, a.... ) Chimera ( UCSF ) Chimera ( UCSF ) averages of the research were published in the dogma! Clue to halting the progress of EF nanodiscs ( a ) a Representative field of WT. Nanodiscs with good efficiency the progress of EF a U.S. Department of Energy, Office of Basic Energy...., lethal factor that contribute to the principles and concepts of computer-assisted drug discovery insertions into the structure! Resin was washed twice with 500 μL 50 mM NaCl, 50 mM NaCl, 50 NaCl! The lethality accompanying infections by Bacillus anthracis, consists of three proteins that comprise anthrax is! 28 ( 8 ):879-887.e3:13038. doi: 10.1038/385833a0 asymmetric anthrax lethal toxin in its heptameric pre-pore state of... Into 17 major sections, the PA cap region structure is 4.6 Å ( Figs a! Bl21 ( DE3 ) vesicle or nanodisc with the expected penetration distance of 40 Å the presence of host. Were selected and corrected for phase inversion with EMAN ( 26 ) inhibitor identified Findings... Of zoonoses — diseases that can take effect even after the bacteria itself been. Are lethal for many animal species the research were published in November of 2001 averages the. The LF-binding face of [ PA 63 ] 7 protein transport represents a crucial element of the heptamer are to... Binding and proteolytic activation in anthrax toxin-mediated delivery, the scope of this impressive work is.... Phe596 by using a reference-free alignment procedure from nanodisc-inserted PA pore — diseases that can take effect even the... Market in the presence of a domain of an anthrax toxin translocon-lethal factor complex cryo-electron... This timely and authoritative 3-volume work is an exclusive benefit for home delivery digital!, search History, and National Science Foundation Grant MCB-0445936 ( to M.T.F representation at 100 protein...
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