Role of the Protective Antigen Octamer in the Molecular Mechanism of Anthrax Lethal Toxin Stabilization in Plasma |
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Authors: | Alexander F Kintzer Iok I Tang Andrew J Miles Evan R Williams Bryan A Krantz |
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Institution: | 1Department of Chemistry, University of California, Berkeley, CA 94720, USA 2Department of Crystallography, Birkbeck College, University of London, London WC1E 7HX, UK 3California Institute for Quantitative Biomedical Research (QB3), University of California, Berkeley, CA 94720, USA 4Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA |
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Abstract: | Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-γ-D-glutamic acid capsule. Atx is comprised of three proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT can assemble on host cell surfaces or extracellularly in plasma. We show that, under physiological conditions in bovine plasma, LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel-forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration, which allows them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that could circulate freely in the blood. |
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Keywords: | Atx anthrax toxin PA protective antigen nPA nicked protective antigen LF lethal factor EF edema factor LT lethal toxin γ-DPGA poly-γ-d-glutamic acid L-capsule lower molecular mass capsule PLB planar lipid bilayer SRCD synchrotron radiation circular dichroism sANTXR2 soluble domain from human anthrax receptor domain nano-ESI nanoelectrospray ionization nanoESI-MS nanoelectrospray ionization mass spectrometry |
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