In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 100, No. 24 ( 2003-11-25), p. 13803-13808
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 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 PA 63 . A majority ( 22 ) were in domain 2, the pore-forming domain. Seven of the domain-2 mutations, located in or adjacent to the 2β 6 strand, the 2β 7 strand, and the 2β 10 -2β 11 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 2β 6 and 2β 7 strands and the 2β 10 –2β 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.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.2436299100
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2003
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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