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

Pedro Jacquez, Gustavo Avila, Kyle Boone, Agamyrat Altiyev, Jens Puschhof, Roland Sauter, Emma Arigi, Blanca Ruiz, Xiuli Peng, Igor Almeida, Michael Sherman, Chuan Xiao, Jianjun Sun

Research output: Contribution to journalArticle

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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 vonWillebrand 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.

Original languageEnglish (US)
Article numbere0130832
JournalPLoS One
Volume10
Issue number6
DOIs
StatePublished - Jun 24 2015

Fingerprint

disulfide bonds
Disulfides
immunoglobulins
Immunoglobulins
antigens
Membranes
Antigens
receptors
sulfides
Fluorescence
Anthrax
fluorescence
Antigen Receptors
Clamping devices
anthrax
anthrax toxin
anthrax toxin receptors
Immunoglobulin Domains
Liposomes
Oxidation-Reduction

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

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. / Jacquez, Pedro; Avila, Gustavo; Boone, Kyle; Altiyev, Agamyrat; Puschhof, Jens; Sauter, Roland; Arigi, Emma; Ruiz, Blanca; Peng, Xiuli; Almeida, Igor; Sherman, Michael; Xiao, Chuan; Sun, Jianjun.

In: PLoS One, Vol. 10, No. 6, e0130832, 24.06.2015.

Research output: Contribution to journalArticle

Jacquez, P, Avila, G, Boone, K, Altiyev, A, Puschhof, J, Sauter, R, Arigi, E, Ruiz, B, Peng, X, Almeida, I, Sherman, M, Xiao, C & Sun, J 2015, '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', PLoS One, vol. 10, no. 6, e0130832. https://doi.org/10.1371/journal.pone.0130832
Jacquez, Pedro ; Avila, Gustavo ; Boone, Kyle ; Altiyev, Agamyrat ; Puschhof, Jens ; Sauter, Roland ; Arigi, Emma ; Ruiz, Blanca ; Peng, Xiuli ; Almeida, Igor ; Sherman, Michael ; Xiao, Chuan ; Sun, Jianjun. / 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. In: PLoS One. 2015 ; Vol. 10, No. 6.
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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 vonWillebrand 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.",
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AU - Altiyev, Agamyrat

AU - Puschhof, Jens

AU - Sauter, Roland

AU - Arigi, Emma

AU - Ruiz, Blanca

AU - Peng, Xiuli

AU - Almeida, Igor

AU - Sherman, Michael

AU - Xiao, Chuan

AU - Sun, Jianjun

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