Identification of residues lining the anthrax protective antigen channel.
In its activated 63 kDa form, the protective antigen (PA) component of anthrax toxin forms a heptameric prepore, which converts to a pore (channel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol. It has been proposed that the prepore-to-pore conversion involves a conformational rearrangement of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-stranded beta barrel. To test this model, we generated Cys substitutions in 24 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductance after adding the thiol-reactive, bilayer-impermeant reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) to the trans compartment. The rationale for these experiments is that reaction of MTS-ET with a Cys residue adds a positively charged group and therefore would likely reduce channel conductance if the residue were in the ion-conducting pathway. We found alternating reduction and absence of reduction of conductance in consecutive residues over two stretches (residues 302-311 and 316-325). This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen and into the bilayer, respectively. Residues connecting these two stretches (residues 312-315) were responsive to MTS-ET, consistent with their being in a turn region. Single channels formed by selected mutants (H304C and N306C) showed multiple conductance step changes in response to MTS-ET, consistent with an oligomeric pore. We also found that the binding site for the channel-blocking tetraalkylammonium ions is located cis relative to the inserted D2L2 loops. These findings constitute strong evidence in favor of the model of conversion of the prepore to a 14-stranded beta barrel pore and solidify the foundation for studies to understand the mechanism of translocation by anthrax toxin.
Benson
E L
EL
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Huynh
P D
PD
Finkelstein
A
A
Collier
R J
RJ
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000187:drug effects; D001408:Bacillus anthracis / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry*; D003902:Detergents; D004553:Electric Conductivity; D007202:Indicators and Reagents; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000187:drug effects; D008698:Mesylates / Q000494:pharmacology; D008969:Molecular Sequence Data; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D013237:Stereoisomerism
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D003902:Detergents; D007202:Indicators and Reagents; D007473:Ion Channels; D008698:Mesylates; D000644:Quaternary Ammonium Compounds; C030325:anthrax toxin; C109153:(2-sulfonatoethyl)methanethiosulfonate; C009405:tetrabutylammonium