Anthrax lethal toxin co-complexes are stabilized by contacts between adjacent lethal factors

The Rockefeller University Press $30.00 J. Gen. Physiol. 2016 Vol. 148 No. 4 273–275 www.jgp.org/cgi/doi/10.1085/jgp.201611681 273 Anthrax toxin is a three-protein toxin that must first assemble before carrying out its physiological function of menacing its eukaryotic host. Much has been done, therefore, to study its assembly both in vitro and on cell surfaces. The three proteins that comprise the toxin are protective antigen (PA), lethal factor (LF), and edema factor (EF). Individually, these proteins are nontoxic, but in combination, they produce toxic complexes (Fig. 1). PA plus LF makes lethal toxin and PA plus EF makes edema toxin. To assemble, PA is first nicked by a protease to yield a 20-kD fragment and the 63-kD fragment, PA63 (Blaustein et al., 1989). PA63 fragments then self-assemble into a ring-shaped prechannel heptamer (Milne et al., 1994; Petosa et al., 1997) or octamer (Kintzer et al., 2009), which can bind to either three or four copies of LF/EF, respectively. The toxin complexes are then endocytosed into endosomes that acidify as they mature, causing the prechannel PA oligomer to convert into a membrane-inserted channel. LF and EF then unfold and translocate through this channel into the cytosol of the host cell. There in the cytosol, LF and EF carry out their physiological functions, catalyzing reactions that disrupt the cell. In this issue, Fabre et al. report a new cryo–electron microscopy (cryo-EM) structure of an anthrax lethal toxin co-complex, containing three copies of LF bound to the PA heptamer (PA7–LF3 complex). This new structure adds another level of complexity to our understanding of anthrax toxin assembly. Although the current study describes a PA7–LF complex, a previous structure of PA in complex with a fragment of LF (LFN) revealed an octameric PA8–LF4 stoichiometry (Fig. 1, inset; Feld et al., 2010). Although both complexes are stable and both complexes are observed on cells (Kintzer et al., 2009), an understanding of which complex predominates during each stage of toxicity is lacking. A novel observation reported in Fabre et al. (2016) is that of interactions between adjacent LF subunits around the ring. Specifically, the C-terminal domains of one LF contact the neighboring LF on its N-terminal domain. The authors speculate that this interaction would stabilize the complex. Because the structure of the octameric complex contained the shortened LFN (and not full-length LF), interactions between LF subunits were not observed (Feld et al., 2010). However, modeling the full-length LF into this complex (based on the new structure by Fabre et al. [2016]) reveals that LFs would similarly make contacts in the octameric complex, and in fact, these contacts would be more extensive than those observed in the heptameric complex. More specifically, the PA7–LF3 co-complex reveals contacts between the first and second and second and third LFs, whereas modeling predicts contacts all the way around the PA oligomer ring in the PA8–LF4 complex. Thus, the octamer would be expected to form a more stable co-complex. How might the presumed stabilization of the lethal toxin co-complex be important to toxicity? Two obvious possibilities are stabilization of the prechannel oligomer and stabilization of the channel co-complex. These are not mutually exclusive, of course. It makes sense that the toxin would want to stabilize itself at the prechannel stage; such stabilization would save the assembled toxin from proteolysis and other potential insults. Studies of the toxin have shown that there exist two potential assembly pathways: one that occurs on the cell surface and another that occurs in solution. The solution assembly pathway favors the octamer over the heptamer in the sense that stability of the prechannel oligomer prevents premature channel formation in solution (Kintzer et al., 2010). Premature channel formation, which happens more readily in heptameric PA complexes with LF at neutral pH and body temperature (Kintzer et al., 2010), leads to aggregation of the membrane-spanning hydrophobic domains and thus heptamers precipitating out of solution to leave the soluble octamers behind. Furthermore, in addition to the avoidance of premature channel formation, the octameric prechannel would have additional LF contacts that could serve to stabilize the prechannel and lessen dissociation of LF from the complex. The presumed stabilization by LF interactions might also be mechanistically important for stabilizAnthrax lethal toxin co-complexes are stabilized by contacts between adjacent lethal factors