Determining the High-Resolution Structures of the Anthrax Toxin Protective Antigen Pore Bound to Its Lethal and Edema Factors

Anthrax toxin, one of the virulence factors of Bacillus anthracis, consists of three individual protein factors: the cell binding/pore-forming protective antigen (PA), and the enzymatic factor lethal factor (LF), and edema factor (EF). Due to its formation of a protein-conducting pore, PA provides an excellent system to study protein translocation. To date, the only structural information available of the PA bound to its enzymatic substrate is of the amino terminal domain of LF, LFn, bound to the pre-pore octamer. This structure has shown that the binding of LF to the PA pre-pore serves to stabilize its unfolding before being translocated through the channel. Using cryo-EM, we determined high-resolution structures of the heptameric PA in its pore conformation bound to LF at 4.6-Å, and bound to EF at 3.6-Å. The PA7LF3 and PA7EF3 pre-pore complexes were inserted into lipid nanodiscs, causing them to take random orientation on cryo-EM grids and allowing us to perform single-particle analysis. The structures contain the pore form PA heptamer bound to a single LF or EF, respectively. The C-terminal domains of LF is disordered in the PA7 LF structure, resulting in no visible electron density. When comparing the LFn domain when bound to PA in the pore state versus the pre-pore complex, there appears to be no significant changes in structure. The first α-helix in LF its unfolded and docked within the α-clamp site. In the structure of PA7EF, the full length of EF protein appears to be ordered, showing domain reorganization within EF upon binding to PA. In addition, the α-clamp is also populated by the first α-helix in EF. These structures can help in our understanding of the mechanisms involved in protein translocation.