Anthrax lethal factor (LF) mediated block of the anthrax protective antigen (PA) ion channel: effect of ionic strength and voltage.
The anthrax toxin complex consists of three different molecules, protective antigen (PA), lethal factor (LF), and edema factor (EF). The activated form of PA, PA(63), forms heptamers that insert at low pH in biological membranes forming ion channels and that are necessary to translocate EF and LF in the cell cytosol. LF and EF are intracellular active enzymes that inhibit the host immune system promoting bacterial outgrowth. Here, PA(63) was reconstituted into artificial lipid bilayer membranes and formed ion-permeable channels. The heptameric PA(63) channel contains a binding site for LF on the cis side of the channel. Full-size LF was found to block the PA(63) channel in a dose- and ionic-strength-dependent way with half-saturation constants in the nanomolar concentration range. The binding curves suggest a 1:1 relationship between (PA(63))(7) and bound LF that blocks the channel. The presence of a His(6) tag at the N-terminal end of LF strongly increases the affinity of LF toward the PA(63) channel, indicating that the interaction between LF and the PA(63) channel occurs at the N terminus of the enzyme. The LF-mediated block of the PA(63)-induced membrane conductance is highly asymmetric with respect to the sign of the applied transmembrane potential. The result suggested that the PA(63) heptamers contain a high-affinity binding site for LF inside domain 1 or the channel vestibule and that the binding is ionic-strength-dependent.
Neumeyer
Tobias
T
Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
Tonello
Fiorella
F
Dal Molin
Federica
F
Schiffler
Bettina
B
Orlik
Frank
F
Benz
Roland
R
D000942:Antigens, Bacterial / Q000378:metabolism* / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000378:metabolism* / Q000494:pharmacology*; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D006639:Histidine / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D008564:Membrane Potentials; D009994:Osmolar Concentration; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007473:Ion Channels; C030325:anthrax toxin; D006639:Histidine