Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 100, Issue 24, Pages 13803-13808Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2436299100
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- NIAID NIH HHS [R01 AI022021, R37 AI022021, AI-22021] Funding Source: Medline
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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 (DIN), were found to coassemble with wild-type PA and generate defective heptameric pore-precursors (prepores). Prepores containing DN-PA 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(63), 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 PA63. A majority (22) were in domain 2, the pore-forming domain. Seven of the domain-2 mutations, located in or adjacent to the 206 strand, the 2beta(7) strand, and the 2beta(10)-2beta(11) loop, gave the DIN phenotype. This study demonstrates the feasibility of high-throughput scanning mutagenesis of a moderate sized protein. The results show that DIN mutations cluster in a single domain and implicate 2beta(6) and 2beta(7) strands and the 2beta(10)-2beta(11) loop in the conformational rearrangement of the prepore to the pore. They also add to the repertoire of mutations available for structure-function studies and for designing new antitoxic agents for treatment of anthrax.
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