Two investigators at OUHSC (Drs. Rod Tweten and Jimmy Ballard) have recently published
studies using high-throughput DNA sequencing and oligonucleotide synthesis (provided by
the OUHSC Laboratory for Genomics and Bioinformatics), to identify potential therapeutic
agents for suppressing the lethal effects of anthrax toxin. 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, Tweten, Ballard and co-workers generated 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) of
the DN mutations were in domain 2, the pore-forming domain. This study demonstrated the
feasibility of high-throughput scanning mutagenesis of a moderate sized protein, accomplished
in a realistic time frame with a reasonable cost. The results show that DN mutations
cluster in a single domain and implicate 26
and 27 strands and the
210 - 211 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. These studies
were recently published in the Proceedings of the National Academy (Mourez, M., M. Yan,
D. B. Lacy, L. Dillon, L. Bentsen, A. Marpoe, C. Maurin, E. Hotze, D. Wigelsworth,
R. A. Pimental, J.D. Ballard, R.J. Collier and R. K. Tweten. 2003. Mapping dominant-negative
mutations of anthrax protective antigen by scanning mutagenesis. Proc. Natl. Acad. Sci.
USA 100:13803-8.)
Two investigators at OUHSC (Drs. Rod Tweten and Jimmy Ballard) have recently published
studies using high-throughput DNA sequencing and oligonucleotide synthesis (provided by
the OUHSC Laboratory for Genomics and Bioinformatics), to identify potential therapeutic
agents for suppressing the lethal effects of anthrax toxin. 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, Tweten, Ballard and co-workers generated 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) of
the DN mutations were in domain 2, the pore-forming domain. This study demonstrated the
feasibility of high-throughput scanning mutagenesis of a moderate sized protein, accomplished
in a realistic time frame with a reasonable cost. The results show that DN mutations
cluster in a single domain and implicate 2
6
and 2