Toxins that modify targets within the cytosol of mammalian cells are crucial virulence factors in anthrax, diphtheria, botulism, cholera and many other bacterial diseases. While all toxins of this class must cross a membrane to reach their intracellular targets, we still do not have a clear understanding of the membrane translocation event for any such toxin. Anthrax toxin translocates its two enzymic moieties, Lethal Factor (LF) and Edema Factor (EF), to the cytosol of mammalian cells through a heptameric pore formed in the endosomal membrane by the Protective Antigen (PA) moiety. We have replicated the translocation event to a good ap- proximation in a model membrane system (planar phospholipid bilayers) and shown that the PA pore acts as an active transporter, rather than simply a water-filled conduit. We identified a crucial structural feature of the pore, dubbed the Phe clamp and formed from the seven Phenylalanine 427 residues, and showed that the pH gradient across the membrane is the primary energy source for translocation. Further, a Brownian ratchet model was put forth to explain translocation in physical chemical terms. In the coming grant period we will study the interaction of the pore with its polypeptide translocation substrates using a variety of approaches. The properties of the N-terminal translocation leader (TL) peptide of LF that are important for initiating translo- cation will be determined, and the location of the leader peptide in the pore will be mapped. We will probe the role of the Phe clamp in translocation and the basis of the dominant-negative phenotype elicited by mutations at Phe427 and other sites in the pore. The structure of the pore will be investigated by electron microscopy and crystallography, and various other approaches will be used to analyze the conformational transition of the prepore to the pore. This research will elucidate a key step in the action of an important bacterial toxin at a level of detail not yet attained with any other toxin;and it will strengthen the foundation for applying toxins to medical applications. PUBLIC HEALTH RELEVANCE: This research will elucidate the structure and mode of action of anthrax toxin, an important bacterial toxin. We will seek an understanding, at a level of detail not yet attained with any other toxin, of how the enzymatic components of anthrax toxin enter mammalian cells via a pore formed in a cellular membrane. As with our past research, these studies are expected to yield findings relevant to uses of toxins in medical applications.