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Probing the pH-dependent prepore to pore transition of Bacillus anthracis protective antigen with differential oxidative protein footprinting.

ABSTRACT: The protective antigen (PA) component of the anthrax toxin (ATx) plays an essential role in the pathogenesis of the bioterrorism bacterium Bacillus anthracis. After oligomerization on the cell surface and docking of lethal factor and/or edema factor, PA is internalized and undergoes a conformational change when exposed to the low pH of the endosome to form a membrane-penetrating pore. While the structure of the PA prepore has been determined, precise structural information regarding the pore state remains lacking. Oxidative protein footprinting (OPF) can provide dynamic structural information about a protein complex through analysis of amino acid oxidation both before and after a conformational change. In this study, PA at pH 7.5 and 5.5 was exposed to hydroxyl radicals generated by ionizing radiation. Mass spectrometry was then used to both identify and quantitate the extent of oxidation of differentially modified residues. Several residues were found to be more readily oxidized at pH 7.5, most of which clustered toward the bottom plane of the prepore heptamer. Two amino acids had greater oxidation rates at pH 5.5, both found on the outer periphery of the prepore. When the OPF results were mapped to a current computational model of the pore, the accessibilities of some residues were consistent with their modeled positions in the pore (i.e., Y688 and V619/I620), while data for other residues (W346 and M350) appeared to conflict with the model. The results from this study illustrate the utility of OPF in generating empirical structural information for yet undetermined structures and offering opportunities for refinement for models thereof.

SUBMITTER: Smedley JG

PROVIDER: S-EPMC2645544 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Probing the pH-dependent prepore to pore transition of Bacillus anthracis protective antigen with differential oxidative protein footprinting.

Smedley James G JG Sharp Joshua S JS Kuhn Jeffrey F JF Tomer Kenneth B KB

Biochemistry 20080912 40

The protective antigen (PA) component of the anthrax toxin (ATx) plays an essential role in the pathogenesis of the bioterrorism bacterium Bacillus anthracis. After oligomerization on the cell surface and docking of lethal factor and/or edema factor, PA is internalized and undergoes a conformational change when exposed to the low pH of the endosome to form a membrane-penetrating pore. While the structure of the PA prepore has been determined, precise structural information regarding the pore sta ...[more]

PMID: 18785752

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Project description:BACKGROUND: Anthrax is caused by Bacillus anthracis that produce two exotoxins, lethal toxin and edema toxin. The lethal toxin is composed of the lethal factor (LF) complexed with the cell binding protective antigen (PA83, 83 kDa). Likewise, the edema factor (EF) binds to the PA83 to form the edema toxin. Once PA83 is bound to the host cell surface, a furin-like protease cleaves the full-length, inactive protein into 63 kDa and 20 kDa antigens (PA63 and PA20). PA63 forms a heptamer and is internalized via receptor mediated endocytosis forming a protease-stable pore, which allows EF and LF to enter the cell and exert their toxic effects.Both proteolytically cleaved protective antigens (PA63 and PA20 fragments) are found in the blood of infected animals. The 63 kDa protective antigen PA63 fragment has been thoroughly studied while little is known about the PA20. METHODS: In this study we examined the role of PA20 using high throughput gene expression analysis of human peripheral blood mononuclear cells (PBMC) exposed to the PA20. We constructed a PA mutant in which a Factor Xa proteolytic recognition site was genetically engineered into the protective antigen PA83 to obtain PA20 using limited digestion of this recombinant PA83 with trypsin. RESULTS: Global gene expression response studies indicated modulation of various immune functions and showed gene patterns indicative of apoptosis via the Fas pathway in a subset of the lymphoid cells. This finding was extended to include observations of increased Caspase-3 enzymatic activity and the identification of increases in the population of apoptotic, but not necrotic cells, based on differential staining methods. We identified a list of approximately 40 inflammatory mediators and heat-shock proteins that were altered similarly upon exposure of PBMC to either rPA20 or B. anthracis spores/vegetative cells. CONCLUSION: This study shows that the PA20 has an effect on human peripheral blood leukocytes and can induce apoptosis in the absence of other PA components.

Dataset Information

Probing the pH-dependent prepore to pore transition of Bacillus anthracis protective antigen with differential oxidative protein footprinting.

Publications

Probing the pH-dependent prepore to pore transition of Bacillus anthracis protective antigen with differential oxidative protein footprinting.

Similar Datasets

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