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¹ Department of Physiology and Biophysics, and Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461
² Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115

Correspondence to Daniel Basilio: dbseyler{at}aecom.yu.edu

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 LF_N (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 LF_N 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 LF_N, through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF_N 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 -clamp, the ring of seven phenylalanines near the channel's entrance.

S.J. Juris's present address is Depts. of Biology and Chemistry, Central Michigan University, Mount Pleasant, MI 48859.

Abbreviations used in this paper: EF, edema factor; LF, lethal factor; MTS-ACE, 2-aminocarbonyl-ethyl-methanethiosulfonate; MTS-ES, 2-sulfonato-ethyl-methanethiosulfonate; MTS-ET, 2-trimethylammonium-ethyl-methanethiosulfonate; PA, protective antigen; PEG, polyethylene-glycol; WT, wild-type.

© 2009 Basilio et al.
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This article has been cited by other articles:

				D. Basilio, S. J. Juris, R. J. Collier, and A. Finkelstein Evidence for a Proton-Protein Symport Mechanism in the Anthrax Toxin Channel J. Cell Biol., February 23, 2009; 184(4): i11 - i11. [Full Text]

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