A major goal in understanding the pathogenesis of the anthrax bacillus is to determine how the protective antigen (PA) pore mediates translocation of the enzymatic components of anthrax toxin across membranes. To obtain structural insights into this mechanism, we constructed PA-pore membrane complexes and visualized them by using negative-stain electron microscopy. Two populations of PA pores were visualized in membranes, vesicle-inserted and nanodisc-inserted, allowing us to reconstruct two virtually identical PA-pore structures at 22-Å resolution. Reconstruction of a domain 4-truncated PA pore inserted into nanodiscs showed that this domain does not significantly influence pore structure. Normal mode flexible fitting of the x-ray crystallographic coordinates of the PA prepore indicated that a prominent flange observed within the pore lumen is formed by the convergence of mobile loops carrying Phe427, a residue known to catalyze protein translocation. Our results have identified the location of a crucial functional element of the PA pore and documented the value of combining nanodisc technology with electron microscopy to examine the structures of membrane-interactive proteins.electron microscopy | protective antigen | normal mode flexible fitting D etermining the structures of the membrane-inserting components of protein toxins that act within cells is critical for understanding how these toxins translocate their enzymatic cargoes across membranes. The three proteins that comprise anthrax toxin combine to form two binary toxins that contribute to the lethality accompanying infections by Bacillus anthracis. Protective antigen (PA), after binding to a cell-surface receptor and being proteolytically activated, oligomerizes to form a heptameric prepore. The prepore binds the enzymatic lethal factor and/or edema factor proteins, and, after the complexes are trafficked to the endosome, the acidic environment induces the prepore to form a pore in the membrane and to translocate the enzymatic moieties to the cytosol (1). A primary goal for understanding this process is to determine the mechanism by which the PA pore functions as a pH gradient-driven translocation machine to transport the enzymatic toxin components across membranes. Even though the x-ray crystallographic structure of the heptameric PA prepore was solved in 1997 (2), a structure of the PA pore has eluded researchers because of aggregation difficulties.In a prior study we used the chaperonin GroEL as a molecular scaffold to avoid this aggregation problem. We showed that the PA pore formed stable complexes with GroEL and solved a structure of a functional PA pore at ∼23-25 Å resolution by using negativestain EM (3). To gain a better structural understanding of this protein translocation machinery, it is imperative that one obtain structures of the pore inserted into membrane bilayers, and we chose a model bilayer system, the apolipoprotein A1-derived lipid nanodisc, for further studies (4). Unlike other systems such as liposomes or detergent micelles, nan...

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