Anthrax Toxin Translocation Complex Reveals insight into the Lethal Factor Unfolding and Refolding Mechanism

Abstract

AbstractTranslocation is essential to the anthrax toxin mechanism. Protective antigen (PA), the translocon component of this AB toxin, forms an oligomeric pore with three key clamp sites that aid in the efficient entry of lethal factor (LF) or edema factor (EF), the enzymatic components of the toxin, into the cell. LF and EF translocate through the PA pore (PApore) with the pH gradient between the endosome and the cytosol facilitating rapid translocation in vivo. 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 novel 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 as well as in the α clamp, the Φ clamp, and the charge clamp. 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.Significance StatementToxins like the anthrax toxin aid bacteria in establishing an infection, evading the immune system, and proliferating inside a host. The anthrax toxin, a proteinaceous AB toxin secreted by Bacillus anthracis, consists of lethal factor and protective antigen. In this work, we explore the molecular details of lethal factor translocation through protective antigen pore necessary for cellular entry. Our cryo electron microscopy results provide evidence of lethal factor secondary structure refolding prior to protective antigen pore exit. Similar to the ribosome exit tunnel, the toxin pore channel likely contributes to native folding of lethal factor. We predict other AB toxins with extended pores also initiate substrate refolding inside the translocon for effective intoxication during bacterial infection, evasion, and proliferation.