Structure of a dominant negative mutant reveals a stalled intermediate state of anthrax protective antigen pore maturation

Anthrax is a severe bacterial infection caused by Bacillus anthracis , which produces a tripartite toxin that includes protective antigen (PA), lethal factor (LF) and edema factor (EF). A series of dominant-negative mutations have been previously identified that prevent the heptameric PA prepore from forming the pH-induced, membrane spanning beta-barrel pore that is required for translocation of EF and LF to the cytoplasm of the infected cell. Here we show that the dominant negative D425A mutation stalls the formation of the pore at a reversible intermediate maturation state, which exhibits many of the structural aspects of the pore but fails to form the phi(ϕ)-clamp and beta-barrel structure needed for full pore maturation. Overall, this structure reveals that ϕ-clamp and beta-barrel pore formation are later steps in the pathway to pore formation, thereby providing a regulatory mechanism to prevent premature translocation of EF and LF.