Characterization of the disulfide bonds in the ectodomain of anthrax toxin receptor 2

Pedro Raymundo Jacquez, University of Texas at El Paso


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. The ectodomain of anthrax toxin receptor 2 (ANTXR2) is composed of a von Willebrand factor A (VWA) domain that binds to anthrax toxin protective antigen (PA) and a newly defined immunoglobulin-like (Ig) domain, in which the disulfide bonds are required for PA pore formation and for the folding of ANTXR2. While the VWA domain has been well characterized, the structure and function of the whole ectodomain (VWA-Ig) are poorly defined, this is mainly due to the limited production of the soluble recombinant protein of the ectodomain. In order to overcome this, the ANTXR2 ectodomain was fused to the C-terminus of bacterial Trigger Factor (TF), a chaperone that mediates the ribosome-associated, co-translational folding of newly synthesized polypeptides in E. coli. Under the control of a cold shock promoter, the fusion protein was overly expressed as a dominant soluble protein at a low temperature in the oxidative cytoplasm of Origami B cells, where formation of the disulfide bonds is favored. Through a series of chromatography, the ANTXR2 ectodomain was purified into homogeneity. The purified ectodomain is functional in binding to PA and mediating PA pore formation on the liposomal membranes, and the yield is applicable for future biochemical and structural characterization. We have also previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig) domain of the anthrax toxin receptor 2 (ANTXR2) inhibited function of the protective antigen (PA) pore, as measured by release of K+ ions from liposomes and from Chinese hamster ovary (CHO) cells, or by the pore-mediated translocation of a model substrate across the plasma membranes. Therefore, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Deletion of 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. Further, 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 Try59, 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. This study elucidates a novel mechanism for anthrax inhibition through modulating receptor disulfide bonds.^

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Recommended Citation

Jacquez, Pedro Raymundo, "Characterization of the disulfide bonds in the ectodomain of anthrax toxin receptor 2" (2014). ETD Collection for University of Texas, El Paso. AAI10118133.