{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T11:02:36Z","timestamp":1790420556220,"version":"4.1.0"},"reference-count":41,"publisher":"National Academy of Sciences","issue":"24","content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2003,11,25]]},"abstract":"<jats:p>\n                    The protective antigen (PA) moiety of anthrax toxin transports edema factor and lethal factor to the cytosol of mammalian cells by a mechanism that depends on its ability to oligomerize and form pores in the endosomal membrane. Previously, some mutated forms of PA, designated dominant negative (DN), were found to coassemble with wild-type PA and generate defective heptameric pore-precursors (prepores). Prepores containing DN\u2013PA are impaired in pore formation and in translocating edema factor and lethal factor across the endosomal membrane. To create a more comprehensive map of sites within PA where a single amino acid replacement can give a DN phenotype, we used automated systems to generate a Cys-replacement mutation for each of the 568 residues of PA\n                    <jats:sub>63<\/jats:sub>\n                    , the active 63-kDa proteolytic fragment of PA. Thirty-three mutations that reduced PA's ability to mediate toxicity at least 100-fold were identified in all four domains of PA\n                    <jats:sub>63<\/jats:sub>\n                    . A majority (\n                    <jats:xref ref-type=\"bibr\">22<\/jats:xref>\n                    ) were in domain 2, the pore-forming domain. Seven of the domain-2 mutations, located in or adjacent to the 2\u03b2\n                    <jats:sub>6<\/jats:sub>\n                    strand, the 2\u03b2\n                    <jats:sub>7<\/jats:sub>\n                    strand, and the 2\u03b2\n                    <jats:sub>10<\/jats:sub>\n                    -2\u03b2\n                    <jats:sub>11<\/jats:sub>\n                    loop, gave the DN phenotype. This study demonstrates the feasibility of high-throughput scanning mutagenesis of a moderate sized protein. The results show that DN mutations cluster in a single domain and implicate 2\u03b2\n                    <jats:sub>6<\/jats:sub>\n                    and 2\u03b2\n                    <jats:sub>7<\/jats:sub>\n                    strands and the 2\u03b2\n                    <jats:sub>10<\/jats:sub>\n                    \u20132\u03b2\n                    <jats:sub>11<\/jats:sub>\n                    loop in the conformational rearrangement of the prepore to the pore. They also add to the repertoire of mutations available for structure\u2013function studies and for designing new antitoxic agents for treatment of anthrax.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2436299100","type":"journal-article","created":{"date-parts":[[2003,11,25]],"date-time":"2003-11-25T15:59:06Z","timestamp":1069775946000},"page":"13803-13808","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":68,"title":["Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis"],"prefix":"10.1073","volume":"100","author":[{"given":"Michael","family":"Mourez","sequence":"first","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73072; and Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, Oklahoma City, OK 73104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming","family":"Yan","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73072; and Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, Oklahoma City, OK 73104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"D. Borden","family":"Lacy","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73072; and Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, Oklahoma City, OK 73104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisa","family":"Dillon","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73072; and Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, Oklahoma City, OK 73104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lori","family":"Bentsen","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73072; and Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, Oklahoma City, OK 73104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy","family":"Marpoe","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73072; and Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, Oklahoma City, OK 73104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cl\u00e9mence","family":"Maurin","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73072; and Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 940 Stanton L. 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