Evidence for a Proton–Protein Symport Mechanism in the Anthrax Toxin Channel

Abstract

The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA63)7, formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA63)7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LFN (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA63)7 channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LFN enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton–protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO3−, which is essentially not titratable, is introduced at most positions in LFN, through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LFN translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA63)7 channel resides at or near its Φ-clamp, the ring of seven phenylalanines near the channel's entrance.

Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen

“…There is evidence that translocation through the PA 63 pore is driven by a transmembrane proton gradient, and we have proposed a Brownian ratchet model of this process that depends on protonation and deprotonation of acidic residues of the translocating polypeptide (21,23,24,36). The pore is known to be strongly cation-selective, reflecting an electrostatic barrier that discriminates against the passage of negatively charged ions.…”

“…Thus, ratcheting of a polypeptide through the pore from the endosome to the cytosol would be predicted, based simply on the proton gradient, the electrostatic barrier within the pore, and Brownian motion. This model has received support from demonstration that an essentially nontitratable anionic group (SO 3 Ϫ ) introduced at any of a variety of locations within LF N serves as a stop-translocation signal (36).…”

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

“…The process begins when the B-component (83 kDa protective antigen PA 83 protein) binds to receptors on cell membranes [1][2][3] and is cleaved by a furin-type protease on the cell surface. The 63 kg/mol fragment, PA 63 , remains receptor bound 4,5 and forms a nanometer-scale heptameric pre-pore, 6 (PA 63 ) 7 , that binds up to three A-components, the 90 kDa lethal factor (LF), or the 89 kDa edema factor (EF) molecules.…”

Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes

“…This configuration could, at best, accommodate extendedchain but not α-helical structure. The narrow ϕ-clamp structure may, at first glance, support the model that LF/EF translocate as fully extended chain (17) via a ΔpH-driven Brownian ratchet mechanism (10,(18)(19)(20)(21)(22)(23). However, a structure of octameric PA (PA 8 ) bound to four PA-binding domains from LF (LF N ) showed an α-helix unfolded from each LF N and docked into an α-clamp cleft (9).…”

“…Between two plausible models, namely, the extended-chain (17) Brownian ratchet model (10,(18)(19)(20)(21)(22)(23) (Fig. 4A) and the allosteric helix-compression model (8) (Fig.…”