title: 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.
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.
authors.affiliation: Department of Biological Sciences & Border Biomedical Research Center, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Pedro
authors.initials: P
authors.lastname: Jacquez
authors.affiliation: Department of Chemistry, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Avila
authors.affiliation: Bioinformatics Program of University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Kyle
authors.initials: K
authors.lastname: Boone
authors.affiliation: Bioinformatics Program of University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Agamyrat
authors.initials: A
authors.lastname: Altiyev
authors.affiliation: Department of Biological Sciences & Border Biomedical Research Center, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Jens
authors.initials: J
authors.lastname: Puschhof
authors.affiliation: Department of Biological Sciences & Border Biomedical Research Center, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Roland
authors.initials: R
authors.lastname: Sauter
authors.affiliation: Department of Biological Sciences & Border Biomedical Research Center, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Emma
authors.initials: E
authors.lastname: Arigi
authors.affiliation: Department of Biological Sciences & Border Biomedical Research Center, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Blanca
authors.initials: B
authors.lastname: Ruiz
authors.affiliation: China National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, P. R. China.
authors.forename: Xiuli
authors.initials: X
authors.lastname: Peng
authors.affiliation: Department of Biological Sciences & Border Biomedical Research Center, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Igor
authors.initials: I
authors.lastname: Almeida
authors.affiliation: Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas, 77555, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Sherman
authors.affiliation: Department of Chemistry, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Chuan
authors.initials: C
authors.lastname: Xiao
authors.affiliation: Department of Biological Sciences & Border Biomedical Research Center, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, 79968, United States of America.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Sun
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D054884:Host-Pathogen Interactions; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007136:Immunoglobulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C447150:ANTXR2 protein, human; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004220:Disulfides; D007136:Immunoglobulins; D008081:Liposomes; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C030325:anthrax toxin; D003545:Cysteine