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Published on: May 16, 2017
Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis
Michael Mourez1, Ming Yan, D Borden Lacy
1Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
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.
Insights
This study mapped mutations in anthrax protective antigen (PA) that create dominant-negative (DN) variants, impairing toxin pore formation. These findings highlight key structural regions involved in anthrax toxin pore assembly and function.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Anthrax toxin's protective antigen (PA) facilitates intracellular delivery of lethal factor and edema factor.
- Dominant-negative (DN) PA mutants disrupt wild-type PA oligomerization and pore formation.
- Understanding PA's structure-function relationship is crucial for developing antitoxin strategies.
Purpose of the Study:
- To comprehensively map residues in PA63 that confer a dominant-negative phenotype upon single amino acid substitution.
- To identify specific structural domains and regions critical for PA pore formation and function.
Main Methods:
- High-throughput scanning mutagenesis was employed, generating Cys-replacement mutations for all 568 residues of PA63.
- Mutant PA proteins were assessed for their ability to mediate anthrax toxin-induced cell toxicity.
- Mutations reducing toxicity by at least 100-fold were identified and characterized.
Main Results:
- Thirty-three mutations conferring a significant reduction in PA-mediated toxicity were identified across all four PA63 domains.
- A majority of these mutations (22) were located in domain 2, the pore-forming domain.
- Seven domain-2 mutations, specifically in or near the 2beta6 strand, 2beta7 strand, and the 2beta10-2beta11 loop, exhibited the dominant-negative phenotype.
Conclusions:
- High-throughput scanning mutagenesis is feasible for large protein analysis.
- Dominant-negative mutations in PA predominantly cluster in domain 2, implicating specific structural elements in the prepore-to-pore transition.
- These findings provide valuable insights for structure-function studies and the design of novel anthrax antitoxins.
