Anthrax protective antigen: Prepore-to-pore conversion

PA(63), the active 63 kDa form of anthrax protective antigen, forms a hepta meric ring-shaped oligomer that is believed to represent a precursor of the membrane pore formed by this protein. When maintained at pH greater than o r equal to 8.0, this "prepore" dissociated to monomeric subunits upon treat ment with SDS at room temperature, but treatment at pH less than or equal t o 7 (or with beta-octylglucoside at pH 8.0) caused it to convert to an SDS- resistant pore-like form. Transition to this form involved major changes in the conformation of loop 2 of domain 2 (D2L2), as evidenced by (i) occlusi on of a chymotrypsin site within D2L2 and (ii) excimer formation by pyrene groups linked to N306C within this loop. The pore-like form retained the ca pacity to bind anthrax toxin A moieties and cell surface receptors, but was unable to form pores in membranes or mediate translocation. Mutant PA(63) in which D2L2 had been deleted was inactive in pore formation and transloca tion but, like the prepore, was capable of forming heptamers that converted to an SDS-resistant form under acidic conditions. Our findings support a m odel of pore formation in which the D2L2 loops move to the membrane-proxima l face of the heptamer and interact to form a 14-strand transmembrane beta- barrel. Concomitantly, domain 2 undergoes a major conformational rearrangem ent, independent of D2L2, that renders the heptamer resistant to dissociati on by SDS. These results provide a basis for further exploration of the rol e of PA(63) in translocation of anthrax toxin's enzymic moieties across mem branes.