Abstract
Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig) domain of the anthrax toxin receptor 2 (ANTXR2) inhibited the function of the protective antigen (PA) pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA) domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax.
Highlights
In order to overcome the host defense system, pathogenic bacteria deliver toxins into the cytoplasm of host cells and disrupt the key cellular metabolic pathways
To determine the exact disulfide pairing in the Ig domain, we generated a series of mutations by replacing Cys with Ala two at a time in all possible combinations: C230A/C255A, C230A/C279A, C230A/C315A, C255A/C279A, C255A/C315A, C279A/C315A
While all of the proteins were expressed at a similar level in E. coli, only the proteins with mutations C230A/ C315A, C255A/C279A and 4C/A were purified as soluble monomeric proteins (Fig 1B and 1C)
Summary
In order to overcome the host defense system, pathogenic bacteria deliver toxins into the cytoplasm of host cells and disrupt the key cellular metabolic pathways. One of the major virulence factors of Bacillus anthracis, is a tripartite A-B toxin It is composed of two catalytic moieties, edema factor (EF) and lethal factor (LF), and a receptor-binding/pore-forming moiety, protective antigen (PA) [2]. The toxin-receptor complexes are internalized by receptor-mediated endocytosis, and the prepore undergoes an acidic pH-dependent conformational rearrangement within the endosome to form a cation-selective, transmembrane pore [7,8]. The PA pore mediates translocation of EF and LF across the endosomal membrane into the cytosol, where EF, an 89-kDa calmodulin-dependent adenylate cyclase, elevates levels of cAMP [9], and LF, a 90-kDa zinc protease, inactivates mitogen-activated protein kinase kinases [10]
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