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.

Fig. 1.

Mapping the 33 defective mutations on the structure of PA₆₃. (Left) A monomeric subunit of the PA₆₃ prepore heptamer is shown with domain 1′ and the calcium ions in dark blue, domain 2 in light blue, domain 3 in yellow, and domain 4 in green. The 33 residues for which a mutated PA was defective yet expressed are colored red. (Right) The mutants with a DN effect are mapped onto domain 2 and indicated in red. The mutants map to three key structural elements indicated in yellow: the 2β₆ strand (T380 and S382), the 2β₇ strand (T393 and N399), and the 2β₁₀–2β₁₁ loop (N422). D425 and F427, also shown in the 2β₁₀–2β₁₁ loop, were identified as sites of DN mutants (28).

Fig. 2.

DN effect of some PA mutants. CHO-K1 cells were incubated with 2 × 10^–10 M PA and 10^–9 M LF_NDTA in the presence of PA mutants or more wild-type PA at various mutant/wild-type PA ratios. The intoxication proceeded for 4 h, at which time protein synthesis was evaluated as described (30). The PA molecules tested are D425K/K397D (□), wild type (○), I364C (⋄), S382C (▵), N399C (▪), T393C (•), T380C (×), N422C(▴), and Y411C (♦).