Abstract
Translocation is essential to the anthrax toxin mechanism. Protective antigen (PA), the binding component of this AB toxin, forms an oligomeric pore that translocates lethal factor (LF) or edema factor, the active components of the toxin, into the cell. Structural details of the translocation process have remained elusive despite their biological importance. To overcome the technical challenges of studying translocation intermediates, we developed a method to immobilize, transition, and stabilize anthrax toxin to mimic important physiological steps in the intoxication process. Here, we report a cryoEM snapshot of PApore translocating the N-terminal domain of LF (LFN). The resulting 3.3 Å structure of the complex shows density of partially unfolded LFN near the canonical PApore binding site. Interestingly, we also observe density consistent with an α helix emerging from the 100 Å β barrel channel suggesting LF secondary structural elements begin to refold in the pore channel. We conclude the anthrax toxin β barrel aids in efficient folding of its enzymatic payload prior to channel exit. Our hypothesized refolding mechanism has broader implications for pore length of other protein translocating toxins.
Highlights
Translocation is essential to the anthrax toxin mechanism
Previous structural studies have generally used urea to avoid aggregation during the transition from PAprepore to PApore[17,18,19,20,21,22]. These approaches have limitations in that they do not account for the low pH electrostatic microenvironment in the pore lumen predicted to be important for lethal factor (LF)-Protective antigen (PA) interactions[23] and they assume similar outcomes for chaotrope and acid induced unfolding
CryoEM density consistent with nascent polypeptide chain translocating the length of P Apore
Summary
Translocation is essential to the anthrax toxin mechanism. Protective antigen (PA), the binding component of this AB toxin, forms an oligomeric pore that translocates lethal factor (LF) or edema factor, the active components of the toxin, into the cell. The resulting 3.3 Å structure of the complex shows density of partially unfolded LFN near the canonical PApore binding site. Our hypothesized refolding mechanism has broader implications for pore length of other protein translocating toxins. We developed an approach to elucidate the structural and mechanistic details of the anthrax toxin during translocation in an effort to understand how LF unfolds in the endosome, translocates through PA, and refolds in the cytosol. A crystal structure of the N-terminal domain of LF (LFN) bound to the PAprepore revealed the α clamp binding site[11]. Blocking of this α clamp binding site results in reduced
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