LAUSR

The lethality of anthrax, a zoonotic disease and bioterrorism agent, is due to the anthrax toxin. This tripartitate toxin consists of protective antigen (PA), lethal factor (LF), and edema factor (EF). The 440 kDa heptameric PA prepore binds up to three molecules of LF, a mitogen-activated protein kinase kinase protease, and/or EF, an adenylate cyclase. This entire complex, bound to receptor proteins, is endocytosed. As the endosome acidifies to pH 5.0 (late endosome), the receptor bound PA prepore unfolds and refolds into an extend beta-barrel pore structure that penetrates the endosomal membrane. This pore unfolds and translocates the 90 kDa LF enzyme across the pH gradient (endosomal pH 5.0 to cytosolic pH 7.0) of the endosomal membrane through the narrow pore lumen in a pH driven hypothesized Brownian ratchet mechanism [1]. The translocated LF then refolds on the cytosolic side of the membrane and disrupts cell signaling resulting in cell lethality. To initiate translocation, the Nterminal region of LF moves away from the main body of LF and binds to the alpha clamp region of the PA pore cap [2]. This initial engagement positions the lysine rich N-terminal portion of LF to interact with the 7-fold phe-clamp located further down the PA pore. The directional translocation of LF depends on protonation of acidic residues, the electrostatic character of the PA pore lumen, and remaining positive charges. Subsequent deprotonation of the translocating peptide after the polypeptide passes the phe-clamp region prevents back transfer. To define the nature of the initial engagement and phe-clamp channel blocking steps that occur at different pHs we are using single particle cryoEM.