TCDB is operated by the Saier Lab Bioinformatics Group

1.C.42 The Channel Forming Bacillus anthracis Protective Antigen (BAPA) Family

The BAPA family is named after the protective antigen (PA) of Bacillus anthracis which is required for receptor binding and entry of anthrax toxin into mammalian cells. The crystal structure of PA has been determined with and without its host receptor CMG2 (Petosa et al., 1997; Santelli et al., 2004). The toxin consists of three protein components; the other two components are the lethal factor (LF) and the edema factor (EF). PA plus LF forms the lethal toxin while PA plus EF forms the edema toxin. PA binds to carbohydrate containing receptors, TEM8 and CMG2, and is cleaved at the sequence RKKR by cell surface proteases yielding the N-terminal PA20 and the C-terminal 63 kDa PA63 which binds LF or EF. Unless PA20 is removed by proteolysis, PA63 is inactive in binding EF and LF and in channel formation. The complex is internalized by endocytosis. Acidification of the vesicles promotes membrane insertion of PA63 before forming heptameric cation selective channels that allow entry of the toxin into the animal cell. Channel formation has been demonstrated in liposomes. Cation transport is partially blocked by LF or EF binding. PA also induces immunity to infection by B. anthrax. Binding of EF (a calmodulin-dependent adenylate cyclase) to the heptameric 63 kDa PA channels in liposomes blocks channel activity only when both are added to the cis side (Neumeyer et al., 2006). Basilio et al. (2011) provided evidence that lethal factor (LF) passes through the protective antigen (PA) channel in an open, unfolded configuration. The translocation process has been reviewed (Thoren and Krantz, 2011).

Homologues include the Clostridium botulinum C2 toxin component C2II which allows entry into mammalian cells of the C2I actin-ADP ribosylating component by a similar mechanism and the Clostridium perfringens iota toxin Ib which allows entry into mammalian cells of the Ia component (Barth et al., 2000; Blöcker et al., 2003). The multistep process for entry of C2 toxin includes (1) activation of C2II by trypsin cleavage, (2) heptamerization, (3) binding of C2IIa oligomers to the carbohydrate receptor on the cell surface, (4) assembly with C2I, (5) receptor-mediated endocytosis of both C2 components into endosomes, and (6) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.

C2II forms voltage-gated cation selective channels (Knapp et al., 2002). Residues 303-331 in C2II are amphipathic with alternating hydrophilic and hydrophobic residues. It probably forms two antiparallel β-strands. Glutamate 307 in the center of the membrane plays a role in the cation selectivity and determines the voltage dependency but is not essential for transport of C2I (Blöcker et al., 2003). Iota toxin Ib reconstituted in artificial membranes has a small single-channel conductance, forming a water-filled general diffusion pore with 6x higher permeability to K+ than Cl-.

As noted above, the protective antigen (PA) component of anthrax toxin forms a homoheptameric pore in the endosomal membrane, creating a narrow passageway for the enzymatic components of the toxin to enter the cytosol. During conversion of the heptameric precursor to the pore, the seven phenylalanine-427 residues converged with the lumen, generating a radially symmetric heptad of solvent-exposed aromatic rings. This 'phi-clamp' structure was required for protein translocation and comprised the major conductance-blocking site for hydrophobic drugs and model cations. The phi-clamp serves a chaperone-like function, interacting with hydrophobic sequences presented by the protein substrate as it unfolds during translocation (Krantz et al., 2005). Models for insertion into the endosomal membrane and entry of the toxin into the cytoplasm have been presented (Puhar and Montecucco, 2007). Phenylalanine-427 of anthrax PA functions in both pore formation and protein translocation (Sun et al., 2008).

Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. The side chains of Phe(427) residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes translocation. Janowiak et al. (2010) described the effects of replacing Phe(427) in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >1,000-fold and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment (Janowiak et al., 2010). 

Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis. Protective antigen forms oligomeric prepores that undergo conversion to membrane-spanning pores by endosomal acidification, and these pores translocate the enzymes lethal factor and oedema factor into the cytosol of target cells. A phi (Φ)-clamp composed of phenylalanine (Phe)427 residues catalyses protein translocation via a charge-state-dependent Brownian ratchet. Protective antigen senses low pH, converts to active pore, and translocates lethal factor and oedema factor. By cryo-electron microscopy with direct electron counting, Jiang et al. 2015 determined the protective antigen pore structure at 2.9-Å resolution. The structure reveals the long-sought-after catalytic Φ-clamp and the membrane-spanning translocation channel, and supports the Brownian ratchet model for protein translocation. Comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi-step mechanism by which low pH is sensed and the membrane-spanning channel is formed.

The generalized transport reaction catalyzed by members of the BAPA family is:

Protein toxin and small molecules (out) protein toxin and small molecules (in)

References associated with 1.C.42 family:

Barth, H., D. Blöcker, J. Behlke, W. Bergsma-Schutter, A. Brisson, R. Benz, and K. Aktories. (2000). Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification. J. Biol. Chem. 275: 18704-18711. 10749859
Basilio, D., L.D. Jennings-Antipov, K.S. Jakes, and A. Finkelstein. (2011). Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins. J Gen Physiol 137: 343-356. 21402886
Bhatnagar, R. and S. Batra. (2001). Anthrax toxin. Crit. Rev. Microbiol. 27: 167-200. 11596878
Blöcker, D., C. Bachmeyer, R. Benz, K. Aktories, and H. Barth. (2003). Channel formation by the binding component of Clostridium botulinum C2 toxin: glutamate 307 of C2II affects channel properties in vitro and pH-dependent C2I translocation in vivo. Biochemistry 42: 5368-5377. 12731878
Blanke, S.R. (2006). Portals and pathways: principles of bacterial toxin entry into host cells. Microbe 1: 26-31.
Bronnhuber A., Maier E., Riedl Z., Hajos G., Benz R. and Barth H. (2014). Inhibitions of the translocation pore of Clostridium botulinum C2 toxin by tailored azolopyridinium salts protects human cells from intoxication. Toxicology. 316:25-33. 24394545
Colby JM. and Krantz BA. (2015). Peptide Probes Reveal a Hydrophobic Steric Ratchet in the Anthrax Toxin Protective Antigen Translocase. J Mol Biol. 427(22):3598-606. 26363343
Förstner, P., F. Bayer, N. Kalu, S. Felsen, C. Förtsch, A. Aloufi, D.Y. Ng, T. Weil, E.M. Nestorovich, and H. Barth. (2014). Cationic PAMAM dendrimers as pore-blocking binary toxin inhibitors. Biomacromolecules 15: 2461-2474. 24954629
Gogol EP., Akkaladevi N., Szerszen L., Mukherjee S., Chollet-Hinton L., Katayama H., Pentelute BL., Collier RJ. and Fisher MT. (2013). Three dimensional structure of the anthrax toxin translocon-lethal factor complex by cryo-electron microscopy. Protein Sci. 22(5):586-94. 23494942
Janowiak, B.E., A. Fischer, and R.J. Collier. (2010). Effects of introducing a single charged residue into the phenylalanine clamp of multimeric anthrax protective antigen. J. Biol. Chem. 285: 8130-8137. 20061382
Jiang, J., B.L. Pentelute, R.J. Collier, and Z.H. Zhou. (2015). Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature 521: 545-549. 25778700
Knapp, O., R. Benz, M. Gibert, J.C. Marvaud, and M.R. Popoff. (2002). Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia. J. Biol. Chem. 277: 6143-6152. 11741922
Krantz, B.A., A. Finkelstein, and R.J. Collier. (2006). Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient. J. Mol. Biol. 355: 968-979. 16343527
Krantz, B.A., R.A. Melnyk, S. Zhang, S.J. Juris, D.B. Lacy, Z. Wu, A. Finkelstein, and R.J. Collier. (2005). A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore. Science 309: 777-781. 16051798
Li, M., S. Chen, Y. Wang, S. Zhang, D. Song, R. Tian, J. Geng, and L. Wang. (2022). Label-free high-precise nanopore detection of endopeptidase activity of anthrax lethal factor regulated by diverse conditions. Biosens Bioelectron 219: 114800. [Epub: Ahead of Print] 36274430
Neumeyer, T., F. Tonello, F. Dal Molin, B. Schiffler, and R. Benz. (2006). Anthrax edema factor, voltage-dependent binding to the protective antigen ion channel and comparison to LF binding. J. Biol. Chem. 281: 32335-32343. 16954207
Petosa, C., R.J. Collier, K.R. Klimpel, S.H. Leppla, and R.C. Liddington. (1997). Crystal structure of anthrax toxin protective antigen. Nature 385: 833-838. 9039918
Puhar, A., and C. Montecucco. (2007). Where and how do anthrax toxins exit endosomes to intoxicate host cells? Trends Microbiol. 15: 477-482. 17983750
Santelli, E., L.A. Bankston, S.H. Leppla, and R.C. Liddington. (2004). Crystal structure of a complex between anthrax toxin and its host cell receptor. Nature 430: 905-908. 15243628
Sun, J., A.E. Lang, K. Aktories, and R.J. Collier. (2008). Phenylalanine-427 of anthrax protective antigen functions in both pore formation and protein translocation. Proc. Natl. Acad. Sci. USA 105: 4346-4351. 18334631
Thoren, K.L. and B.A. Krantz. (2011). The unfolding story of anthrax toxin translocation. Mol. Microbiol. 80: 588-595. 21443527
Yamini, G., N. Kalu, and E.M. Nestorovich. (2016). Impact of Dendrimer Terminal Group Chemistry on Blockage of the Anthrax Toxin Channel: A Single Molecule Study. Toxins (Basel) 8:. 27854272
Yannakopoulou, K., L. Jicsinszky, C. Aggelidou, N. Mourtzis, T.M. Robinson, A. Yohannes, E.M. Nestorovich, S.M. Bezrukov, and V.A. Karginov. (2011). Symmetry requirements for effective blocking of pore-forming toxins: comparative study with α-, β-, and γ-cyclodextrin derivatives. Antimicrob. Agents Chemother. 55: 3594-3597. 21555769
Zhang, S., A. Finkelstein, and R.J. Collier. (2004). Evidence that translocation of anthrax toxin''s lethal factor is initiated by entry of its N terminus into the protective antigen channel. Proc. Natl. Acad. Sci. USA 101: 16756-16761. 15548616