Anthrax toxin allows the bacteria to evade the immune system, proliferate, and ultimately kill the host animal.

[2] Research on anthrax toxin also provides insight into the generation of macromolecular assemblies, and on protein translocation, pore formation, endocytosis, and other biochemical processes.

Anthrax is a disease caused by Bacillus anthracis, a spore-forming, Gram positive, rod-shaped bacterium (Fig.

It does this by the formation of pores that span the cell membrane, allowing the entry of the toxin, though the mechanism is not fully understood.

When PA20 dissociates, the remaining receptor-bound portion of PA, called PA63, may assemble into either a heptameric[5] or octameric[6] ring-shaped oligomer.

This ring-shaped oligomer is often referred to as the pre-pore (or pre-channel) form of PA, since later in the pathway it will become a translocase pore (or channel).

EF and LF are driven through the channel by a pH gradient, allowing the enzyme factors to enter the cytosol.

The molecular interactions are apparent upon performing a detailed analysis of the structures of PA, EF, LF, and the cellular receptors (ANTXR1 and ANTXR2).

Analyses on binding sites and conformational changes augmented the structural studies, elucidating the functions of each domain of PA, LF, and EF, as briefly outlined in Table 1.

[12] This specificity of PA and the receptor CMG2 (similar to type I integrins) is due to interactions through a metal ion dependent adhesion site (MIDAS), a hydrophobic groove, and a β-hairpin projection.

[11] The structure they solved was of a non-membrane bound pre-pore, the conformation of the heptamer before the complex extends a β-barrel through the plasma membrane to shuttle the LF and EF into the cytosol.

Heptamerization and pore formation is sterically hindered by the PA20 fragment, but when it is removed from the top of the monomer, the pre-pore is quickly formed.

The heptamer formation causes no major changes in the conformation of each individual monomer, but by coming together, more than 15400 Ų (154 nm2) of protein surface is buried.

[15] Studies on the binding region of LF and EF demonstrated that a large surface area contacts with domain 1 of two adjacent PA63 molecules when in the heptamer conformation.

The co-crystal structure of the PA octamer in complex with N-terminal LF revealed that the binding interaction is, in fact, two discontinuous sites.

[14] One site, termed the C-terminal subsite, resembles a classic "hot-spot" with predicted salt bridges and electrostatic interactions.

The other site, termed the alpha-clamp subsite, is a deep cleft that nonspecifically binds the N-terminal alpha helix and short beta-strand of LF, guiding the N-terminus of the substrate towards the PA prechannel lumen.

In this manner, the alpha clamp aids in protein translocation, nonspecifically binding and subsequently unfolding secondary structure as it unfurls from the substrate.

Dynamin and Eps15 are required for this endocytosis to occur, indicating that anthrax toxin enters the cell via the clathrin-dependent pathway.

Once inside, the complex is transferred to an acidic compartment, where the heptamer, still in the non-membrane-spanning pre-pore conformation, is prepared for translocation of EF and LF into the cytosol.

It is thought that PA acts like these multimeric membrane proteins that form β-barrels made from stretches of both polar and non-polar amino acids from each monomer.

Additional electrophysiological measurements of cysteine substitutions place the amino acids of this loop inside the lumen of the membrane inserted pore.

On artificial bilayers, this occurs when the pH is dropped from 7.4 to 6.5, suggesting that the trigger for insertion involves a titration of histidines.

Furthermore, another histidine is located at the base of the Greek-key motif along with a number of hydrophobic residues (on the green segment in figures 7 and 9a).

[13] Second, the drop in pH causes a disordered loop and a Greek-key motif in the PA domain 2 to fold out of the heptamer pre-pore and insert through the wall of the acidic vesicle, leading to pore formation (Figures 7–9).

Protonation of these histidines causes the domains to separate enough to allow the Greek-key to flop out and help form the β-hairpin involved in insertion.

When a molecule is in this conformation, the N-terminus is freed and drawn into the pore by the proton gradient and positive transmembrane potential.

The interplay between the phenylalanine clamp and the protonation state cause a ratcheting effect that drives the protein though until enough has crossed into the cytoplasm to drag the rest through the pore as the N-terminus refolds.