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  1. Article: Secondary Structure Preferences of the Anthrax Toxin Protective Antigen Translocase

    Das, Debasis / Bryan A. Krantz

    Journal of Molecular Biology. 2017 Mar. 10, v. 429

    2017  

    Abstract: In order for many proteins to move across hydrophobic membrane bilayers, they must be unfolded and translocated by a membrane-embedded channel. These translocase channels interact with the substrate proteins they translocate via hydrophobic pore loops ... ...

    Abstract In order for many proteins to move across hydrophobic membrane bilayers, they must be unfolded and translocated by a membrane-embedded channel. These translocase channels interact with the substrate proteins they translocate via hydrophobic pore loops and cleft structures called clamps. The molecular basis for how clamps facilitate unfolding and translocation is poorly understood. Anthrax toxin is composed of three proteins, a translocase channel-forming subunit, called protective antigen (PA), and two substrate proteins, called lethal factor (LF) and edema factor. Oligomeric PA forms a large channel that contains three types of polypeptide clamp sites: an α clamp, a phenylalanine clamp, and a charge clamp. Currently, it is thought that these clamp sites operate allosterically and promote translocation via an allosteric helix compression mechanism. Here, we report on the substrate secondary structure dependence of the PA channel. Peptides derived from regions of LF with high α-helical content bound cooperatively, but those derived from β-sheet regions in LF did not, suggesting that an allosteric site preferentially recognizes α-helical structure over β-sheet structure. Peptides derived from helical sites in LF showed increasingly longer single-channel blockades as a function of peptide concentration, a result that was consistent with stronger clamping behavior and reduced backsliding. Moreover, peptides derived from helical regions of LF translocated more efficiently than peptides derived from β-sheet regions of LF. Overall, in support of the allosteric helix compression model, we find that the channel prefers α-helical sequences over β-sheet sequences.
    Keywords amino acid motifs ; anthrax toxin ; cell membranes ; hydrophobicity ; membrane proteins ; models ; phenylalanine ; polypeptides
    Language English
    Dates of publication 2017-0310
    Size p. 753-762.
    Publishing place Elsevier Ltd
    Document type Article
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2017.01.015
    Database NAL-Catalogue (AGRICOLA)

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  2. Article ; Online: Atomic structures of anthrax toxin protective antigen channels bound to partially unfolded lethal and edema factors

    Nathan J. Hardenbrook / Shiheng Liu / Kang Zhou / Koyel Ghosal / Z. Hong Zhou / Bryan A. Krantz

    Nature Communications, Vol 11, Iss 1, Pp 1-

    2020  Volume 10

    Abstract: Abstract Following assembly, the anthrax protective antigen (PA) forms an oligomeric translocon that unfolds and translocates either its lethal factor (LF) or edema factor (EF) into the host cell. Here, we report the cryo-EM structures of heptameric PA ... ...

    Abstract Abstract Following assembly, the anthrax protective antigen (PA) forms an oligomeric translocon that unfolds and translocates either its lethal factor (LF) or edema factor (EF) into the host cell. Here, we report the cryo-EM structures of heptameric PA channels with partially unfolded LF and EF at 4.6 and 3.1-Å resolution, respectively. The first α helix and β strand of LF and EF unfold and dock into a deep amphipathic cleft, called the α clamp, which resides at the interface of two PA monomers. The α-clamp-helix interactions exhibit structural plasticity when comparing the structures of lethal and edema toxins. EF undergoes a largescale conformational rearrangement when forming the complex with the channel. A critical loop in the PA binding interface is displaced for about 4 Å, leading to the weakening of the binding interface prior to translocation. These structures provide key insights into the molecular mechanisms of translocation-coupled protein unfolding and translocation.
    Keywords Science ; Q
    Language English
    Publishing date 2020-02-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article: Dynamic Phenylalanine Clamp Interactions Define Single-Channel Polypeptide Translocation through the Anthrax Toxin Protective Antigen Channel

    Ghosal, Koyel / Bryan A. Krantz / Debasis Das / Jennifer M. Colby / Paramjit S. Arora / Stephen T. Joy

    Journal of Molecular Biology. 2017 Mar. 24, v. 429

    2017  

    Abstract: Anthrax toxin is an intracellularly acting toxin where sufficient detail is known about the structure of its channel, allowing for molecular investigations of translocation. The toxin is composed of three proteins, protective antigen (PA), lethal factor ( ...

    Abstract Anthrax toxin is an intracellularly acting toxin where sufficient detail is known about the structure of its channel, allowing for molecular investigations of translocation. The toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxin's translocon, PA, translocates the large enzymes, LF and EF, across the endosomal membrane into the host cell's cytosol. Polypeptide clamps located throughout the PA channel catalyze the translocation of LF and EF. Here, we show that the central peptide clamp, the ϕ clamp, is a dynamic site that governs the overall peptide translocation pathway. Single-channel translocations of a 10-residue, guest–host peptide revealed that there were four states when peptide interacted with the channel. Two of the states had intermediate conductances of 10% and 50% of full conductance. With aromatic guest–host peptides, the 50% conducting intermediate oscillated with the fully blocked state. A Trp guest–host peptide was studied by manipulating its stereochemistry and prenucleating helix formation with a covalent linkage in the place of a hydrogen bond or hydrogen-bond surrogate (HBS). The Trp peptide synthesized with ʟ-amino acids translocated more efficiently than peptides synthesized with D- or alternating D,ʟ-amino acids. HBS stapled Trp peptide exhibited signs of steric hindrance and difficulty translocating. However, when mutant ϕ clamp (F427A) channels were tested, the HBS peptide translocated normally. Overall, peptide translocation is defined by dynamic interactions between the peptide and ϕ clamp. These dynamics require conformational flexibility, such that the peptide productively forms both extended-chain and helical states during translocation.
    Keywords acids ; anthrax toxin ; cytosol ; enzymes ; hydrogen bonding ; mutants ; phenylalanine ; physiological transport ; polypeptides ; proteins ; stereochemistry
    Language English
    Dates of publication 2017-0324
    Size p. 900-910.
    Publishing place Elsevier Ltd
    Document type Article
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2017.02.005
    Database NAL-Catalogue (AGRICOLA)

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    In stock of ZB MED Bonn / Germany

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  4. Article: Role of the α Clamp in the Protein Translocation Mechanism of Anthrax Toxin

    Brown, Michael J / Bryan A. Krantz / Katie L. Thoren

    Journal of Molecular Biology. 2015 Oct. 09, v. 427

    2015  

    Abstract: Membrane-embedded molecular machines are utilized to move water-soluble proteins across these barriers. Anthrax toxin forms one such machine through the self-assembly of its three component proteins—protective antigen (PA), lethal factor, and edema ... ...

    Abstract Membrane-embedded molecular machines are utilized to move water-soluble proteins across these barriers. Anthrax toxin forms one such machine through the self-assembly of its three component proteins—protective antigen (PA), lethal factor, and edema factor. Upon endocytosis into host cells, acidification of the endosome induces PA to form a membrane-inserted channel, which unfolds lethal factor and edema factor and translocates them into the host cytosol. Translocation is driven by the proton motive force, composed of the chemical potential, the proton gradient (ΔpH), and the membrane potential (Δψ). A crystal structure of the lethal toxin core complex revealed an “α clamp” structure that binds to substrate helices nonspecifically. Here, we test the hypothesis that, through the recognition of unfolding helical structure, the α clamp can accelerate the rate of translocation. We produced a synthetic PA mutant in which an α helix was crosslinked into the α clamp to block its function. This synthetic construct impairs translocation by raising a yet uncharacterized translocation barrier shown to be much less force dependent than the known unfolding barrier. We also report that the α clamp more stably binds substrates that can form helices than those, such as polyproline, that cannot. Hence, the α clamp recognizes substrates by a general shape-complementarity mechanism. Substrates that are incapable of forming compact secondary structure (due to the introduction of a polyproline track) are severely deficient for translocation. Therefore, the α clamp and its recognition of helical structure in the translocating substrate play key roles in the molecular mechanism of protein translocation.
    Keywords acidification ; anthrax toxin ; crosslinking ; crystal structure ; cytosol ; endocytosis ; membrane potential ; mutants ; protein transport ; proteins ; proton-motive force
    Language English
    Dates of publication 2015-1009
    Size p. 3340-3349.
    Publishing place Elsevier Ltd
    Document type Article
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2015.08.024
    Database NAL-Catalogue (AGRICOLA)

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    In stock of ZB MED Bonn / Germany

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  5. Article ; Online: Hijacking Multivesicular Bodies Enables Long-Term and Exosome-Mediated Long-Distance Action of Anthrax Toxin

    Laurence Abrami / Lucia Brandi / Mahtab Moayeri / Michael J. Brown / Bryan A. Krantz / Stephen H. Leppla / F. Gisou van der Goot

    Cell Reports, Vol 5, Iss 4, Pp 986-

    2013  Volume 996

    Abstract: Anthrax lethal toxin is a classical AB toxin comprised of two components: protective antigen (PA) and lethal factor (LF). Here, we show that following assembly and endocytosis, PA forms a channel that translocates LF, not only into the cytosol, but also ... ...

    Abstract Anthrax lethal toxin is a classical AB toxin comprised of two components: protective antigen (PA) and lethal factor (LF). Here, we show that following assembly and endocytosis, PA forms a channel that translocates LF, not only into the cytosol, but also into the lumen of endosomal intraluminal vesicles (ILVs). These ILVs can fuse and release LF into the cytosol, where LF can proteolyze and disable host targets. We find that LF can persist in ILVs for days, fully sheltered from proteolytic degradation, both in vitro and in vivo. During this time, ILV-localized LF can be transmitted to daughter cells upon cell division. In addition, LF-containing ILVs can be delivered to the extracellular medium as exosomes. These can deliver LF to the cytosol of naive cells in a manner that is independent of the typical anthrax toxin receptor-mediated trafficking pathway, while being sheltered from neutralizing extracellular factors of the immune system.
    Keywords Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2013-11-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: Anthrax toxin receptor drives protective antigen oligomerization and stabilizes the heptameric and octameric oligomer by a similar mechanism.

    Alexander F Kintzer / Harry J Sterling / Iok I Tang / Evan R Williams / Bryan A Krantz

    PLoS ONE, Vol 5, Iss 11, p e

    2010  Volume 13888

    Abstract: Background Anthrax toxin is comprised of protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins are individually nontoxic; however, when PA assembles with LF and EF, it produces lethal toxin and edema toxin, respectively. ... ...

    Abstract Background Anthrax toxin is comprised of protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins are individually nontoxic; however, when PA assembles with LF and EF, it produces lethal toxin and edema toxin, respectively. Assembly occurs either on cell surfaces or in plasma. In each milieu, PA assembles into a mixture of heptameric and octameric complexes that bind LF and EF. While octameric PA is the predominant form identified in plasma under physiological conditions (pH 7.4, 37°C), heptameric PA is more prevalent on cell surfaces. The difference between these two environments is that the anthrax toxin receptor (ANTXR) binds to PA on cell surfaces. It is known that the extracellular ANTXR domain serves to stabilize toxin complexes containing the PA heptamer by preventing premature PA channel formation--a process that inactivates the toxin. The role of ANTXR in PA oligomerization and in the stabilization of toxin complexes containing octameric PA are not understood. Methodology Using a fluorescence assembly assay, we show that the extracellular ANTXR domain drives PA oligomerization. Moreover, a dimeric ANTXR construct increases the extent of and accelerates the rate of PA assembly relative to a monomeric ANTXR construct. Mass spectrometry analysis shows that heptameric and octameric PA oligomers bind a full stoichiometric complement of ANTXR domains. Electron microscopy and circular dichroism studies reveal that the two different PA oligomers are equally stabilized by ANTXR interactions. Conclusions We propose that PA oligomerization is driven by dimeric ANTXR complexes on cell surfaces. Through their interaction with the ANTXR, toxin complexes containing heptameric and octameric PA oligomers are similarly stabilized. Considering both the relative instability of the PA heptamer and extracellular assembly pathway identified in plasma, we propose a means to regulate the development of toxin gradients around sites of infection during anthrax pathogenesis.
    Keywords Medicine ; R ; Science ; Q
    Subject code 540
    Language English
    Publishing date 2010-11-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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