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

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.

Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile

“…To address this, the high-resolution structure of the translocation complex needs to be clari ed. Recently, the rst attempt to determine the structure of the translocation complex of PA was made 46 .…”

Overview of Bacterial Protein Toxins from Pathogenic Bacteria: Mode of Action and Insights into Evolution

“…The whole assembled toxin is endocytosed in clathrin-coated vesicles. Upon acidification of EEs, the PA prepore inserts into the endosomal membrane, leading to a functional pore that allows the translocation of partially unfolded LF or EF into the cytosol [ 84 , 85 , 86 , 87 , 88 , 89 , 90 ]. The binding components, Ib and C2-II, of C. perfringens iota toxin and C. botulinum C2 toxin, respectively, recognize distinct cell surface receptors ( Table 1 ) and use a similar mechanism as PA to translocate their enzymatic components, Ia and C2-I, respectively, into the cytosol from acidified endocytic vesicles [ 91 , 92 ].…”

Challenges and approaches to studying pore-forming proteins

“…( B ) Membrane mimetic technologies enable structural studies of PFPs in a near-native lipid membrane context. Shown are various examples of nanodiscs [ 15 , 161–163 ], liposomes [ 39 , 163 ], detergent [ 4 ] and amphipols [ 88 , 164 ] which have all been used to study the pore and prepore forms of PFPs. Select cryoEM examples; MPEG1 [ 39 ], XaxAB [ 4 ], Anthrax toxin [ 15 ] and polyC9 [ 88 ] ( C ) Microfluidic and in situ time-resolved cryoEM techniques provide structural insight over small increments of time.…”

“…Broadly, PFPs accomplish biological roles in cell signalling [ 5–7 ], programmed cell death [ 8–10 ], killing of other cells and organisms [ 11 , 12 ], defence, development [ 13 , 14 ], delivery of effector molecules [ 15–18 ], as well as providing a means of digestion. The most obvious function of PFPs — cell killing — is achieved by disrupting the membrane bilayer, resulting in either osmotic shock or diffusion of other effector molecules between membrane compartments.…”