Anthrax toxin translocation complex reveals insight into the lethal factor unfolding and refolding mechanism

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.

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Fig: Anthrax toxin immobilization, translocation, and nanodisc stabilization (TITaNS) method (A) PApore side view slice with funnel shape from α clamp (yellow) to Φ clamp (orange) and charge clamp (red) inside pore β barrel channel indicated. (B) Immobilization of LFN (magenta) PAprepore (blue) complexes on thiol sepharose beads (grey surface). (C) PAprepore transitioned to PApore. (D) Predicted translocation complex of LFN -PApore at low pH. (E) Addition of pre-nanodisc micelle (green) to complex. (F) Nanodisc formation. (G) LFN-PApore-Nanodisc translocation complexes at pH 5.5 on cryoEM grid.

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.

Machen, A.J., Fisher, M.T. & Freudenthal, B.D. Anthrax toxin translocation complex reveals insight into the lethal factor unfolding and refolding mechanism. Sci Rep 11, 13038 (2021). https://doi.org/10.1038/s41598-021-91596-3

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