Evidence for a proton-protein symport mechanism in the anthrax toxin channel.
The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA(63))(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 (PA(63))(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 (PA(63))(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 SO(3)(-), 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 (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.
Basilio
Daniel
D
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dbseyler@aecom.yu.edu
Juris
Stephen J
SJ
Collier
R John
RJ
Finkelstein
Alan
A
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001408:Bacillus anthracis / Q000502:physiology*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics; D017136:Ion Transport / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D021381:Protein Transport
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin