title: Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis.
abstract: 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-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 PA63, 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 2beta6 strand, the 2beta7 strand, and the 2beta10-2beta11 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 2beta6 and 2beta7 strands and the 2beta10-2beta11 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.
authors: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
authors: Michael
authors: M
authors: Mourez
authors: Ming
authors: M
authors: Yan
authors: D Borden
authors: DB
authors: Lacy
authors: Lisa
authors: L
authors: Dillon
authors: Lori
authors: L
authors: Bentsen
authors: Amy
authors: A
authors: Marpoe
authors: Clémence
authors: C
authors: Maurin
authors: Eileen
authors: E
authors: Hotze
authors: Darran
authors: D
authors: Wigelsworth
authors: Ruth-Anne
authors: RA
authors: Pimental
authors: Jimmy D
authors: JD
authors: Ballard
authors: R John
authors: RJ
authors: Collier
authors: Rodney K
authors: RK
authors: Tweten
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity; D001408:Bacillus anthracis / Q000235:genetics* / Q000276:immunology* / Q000472:pathogenicity; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000633:toxicity; D016466:CHO Cells; D006224:Cricetinae; D005798:Genes, Bacterial; D008297:Male; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D010449:Peptide Mapping; D051381:Rats; D011916:Rats, Inbred F344
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin