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 (c hannel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol. It has been proposed tha t the prepore-to-pore conversion involves a conformational rearrangeme nt of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-strande d beta barrel, To test this model, we generated Cys substitutions in 2 4 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductanc e after adding the thiol-reactive, bilayer-impermeant reagent methanet hiosulfonate 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 likel y reduce channel conductance if the residue were in the ion-conducting pathway. We found alternating reduction and absence of reduction of c onductance in consecutive residues over two stretches (residues 302-31 1 and 316-325), This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen an d into the bilayer, respectively. Residues connecting these two stretc hes (residues 312-315) were responsive to MTS-ET, consistent with thei r 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 locat ed 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.