The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore

<div><p>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.</p></div

Homology modeling of the Ig domain and docking of the atomic structure into the reconstructed EM maps.

<p><b>A</b>. The structure of the Ig domain is generated by homologous modeling and grafted to the crystal structure of the VWA domain through energy minimization. The secondary structure is colored as α-helices in orange, β-sheets in purple and loops in grey. The disulfide bonds C39-C218, C255-C279 and C230-C315 are shown as stick models, colored in yellow. Note: Trp59 is shown in a sphere model and labeled. <b>B</b>, <b>D</b>, and <b>F</b> are ribbon diagrams of PA-R318 heptameric complex viewed from top, bottom and side, respectively. <b>C</b>, <b>E</b>, and <b>G</b> are surface rendered density maps from reconstruction of negatively stained PA-R318 heptameric complex, docked with the modeled structure and viewed from top, bottom and side, respectively. Segmented map density of one subunit of the receptor ectodomain is shown on up-right in <b>E</b> or lower-left in <b>G</b>, respectively. The map was rendered at the level of one standard deviation above the average density value of the map. Within the PA heptamer, each of the seven monomers are colored in red, orange, yellow, green, cyan, magenta, and purple, respectively. The VWA domains and the Ig domains are colored in blue and green, respectively. The reconstructed EM maps are rendered in magenta.</p

3D reconstruction of negatively stained PA-TF-R318 and PA-TF-R318(4C/A) detected the disulfide deletion-induced conformational changes on the VWA domain.

<p><b>A</b>, <b>C</b>, and <b>E</b> are surface rendered density maps of PA-TF-R318 heptameric complex viewed from top, bottom and side. <b>B</b>, <b>D</b>, and <b>F</b> are surface rendered density maps of PA-TF-R318(4C/A) viewed from top, bottom and side. The crystal structure of the PA-VWA heptameric complex was docked in the reconstructed maps. <b>G</b> is the side view of the superposed density maps from PA-TF-R318 (transparent grey) and PA-TF-R318(4C/A) (solid green). <b>H</b> is the side view of the superposed density map from PA-TF-R318 (transparent grey) and PA-R318 (solid magenta) showing high similarity of both maps. <b>I</b> and <b>K</b> are the zoom-in monomeric view for the area at the lower part of the complexes as show in <b>G</b> and <b>H</b> respectively. <b>J</b> is the ribbon diagram of the fitted PA and VWA structure at the same orientation and magnification as in <b>I</b> and <b>K</b>. All of the maps were rendered at the level of three and half times standard deviation above the average density value of the maps. The PA is colored in red and the VWA is colored in blue. Trp59 of VWA is rendered as space-filling model and labeled.</p

Compared to PA-R318, PA-R318(C255/279A) exhibited a significant conformational change on the VWA domain.

<p><b>A</b>, <b>B</b>, and <b>C</b> are surface rendered density maps of PA-R318 heptameric complex viewed from top, bottom and side in magenta. <b>E</b>, <b>F</b>, and <b>G</b> are surface rendered density maps of PA-R318(C255/279A) viewed from top, bottom and side in cyan. The crystal structure of the PA-VWA heptameric complex was docked in the reconstructed maps. <b>D</b> is the side view of the superimposed density maps from PA-R318 (transparent magenta) and PA-R318(C255/279A) (solid cyan). <b>H</b> is the side view of the superimposed density map from PA-R318 (transparent magenta) and PA-R318(C255/279A) (solid cyan) that shows the missing densities in the VWA domain. <b>I</b> is the ribbon diagram of the crystal structure of the PA-VWA heptamer at the same orientation and magnification as in <b>H</b>. All of the maps were rendered at the level of three and half times standard deviation above the average density value of the maps. The PA is colored in red and the VWA is colored in blue. Trp59 of VWA is rendered as spherical model and labeled.</p

The residues C255/C279 and C230/C315 form two disulfide bonds in the Ig domain of ANTXR2.

<p><b>A.</b> In the ANTXR2 ectodomain (residues 38–318), C255/C279 and C230/C315 form two disulfide bonds in the Ig domain (residues 219–318). <b>B.</b> The purified TF-R318 and the C/A mutants of TF-R318 as indicated were run in SDS-PAGE, stained by Coomassie blue. <b>C.</b> R318 and the R318 C/A mutants were purified after removal of TF tags and run in SDS-PAGE, followed by Coomassie blue staining.</p

EM Data collection and image processing of negatively stained samples.

<p>EM Data collection and image processing of negatively stained samples.</p

Mutations of C255A/C279A, but not C230A/C315A, resulted in a significant conformational change on the receptor ectodomain.

<p>5 μM of the purified receptor domains were incubated in the 20 mM Tris-HCl (pH 7.3), 100 mM NaCl, in the presence or absence of 5 mM TCEP. The intrinsic Trp59 fluorescence spectrum was measured with excitation at 290 nm and emission at 300–370 nm. Note: two distinct spectrum peaks (320 nm and 330 nm) were detected and represented as two distinct conformations, denoted as C1 and C2.</p