MOLECULAR ARCHITECTURE OF A TOXIN PORE - A 15-RESIDUE SEQUENCE LINES THE TRANSMEMBRANE CHANNEL OF STAPHYLOCOCCAL ALPHA-TOXIN

Staphylococcus aureus alpha-toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores, During assem bly, the formation of a pre-pore precedes membrane permeabilization; t he latter is linked to a conformational change in the oligomer. Here, 41 single-cysteine replacement toxin mutants were thiol-specifically l abelled with the polarity-sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attache d to residues in the sequence 118-140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluo rophore to a hydrophobic environment. Within this region, two function ally distinct parts could be identified. For mutants at positions 126- 140, the shifts were partially reversed after membrane solubilization by detergents, indicating a direct interaction of the label with the m embrane lipids. Membrane insertion of this sequence occurred together with the final prepore to pore transition of the heptamer. Thus residu es 126-140 constitute a transmembrane sequence in the pore, With label led residues 118-124, pre-pore assembly was the critical event to indu ce the spectral shifts, which persisted after the removal of membrane lipids and hence probably reflects protomer-protomer contacts within t he heptamer. Finally, a derivative of the mutant N121C yielded occlude d pores which could be opened by reductive reversal of the modificatio n. Therefore this residue probably lines the lumen of the pore.