Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin.

Journal article

Staphylococcus aureus alpha-toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores. During assembly, the formation of a pre-pore precedes membrane permeabilization; the latter is linked to a conformational change in the oligomer. Here, 41 single-cysteine replacement toxin mutants were thiol-specifically labelled with the polarity-sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attached to residues in the sequence 118-140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluorophore to a hydrophobic environment. Within this region, two functionally 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 membrane lipids. Membrane insertion of this sequence occurred together with the final pre-pore to pore transition of the heptamer. Thus residues 126-140 constitute a transmembrane sequence in the pore. With labelled residues 118-124, pre-pore assembly was the critical event to induce the spectral shifts, which persisted after the removal of membrane lipids and hence probably reflects protomer-protomer contacts within the heptamer. Finally, a derivative of the mutant N121C yielded occluded pores which could be opened by reductive reversal of the modification. Therefore this residue probably lines the lumen of the pore.

Authors: Valeva, A; Weisser, A; Walker, B; Kehoe, M; Bayley, J

• Publication status: Published
• Journal: EMBO journal
• Volume: 15
• Issue: 8
• Pages: 1857-1864
• Publication date: 1996-04-01
• EISSN: 1460-2075
• ISSN: 0261-4189
• Language: English
• Keywords: Mutagenesis, Site-Directed; Molecular Structure; Protein Conformation; Liposomes; Cell Membrane; Binding Sites; Staphylococcus aureus; Spectrometry, Fluorescence; Bacterial Toxins; Amino Acid Sequence; Molecular Sequence Data; 2-Naphthylamine; Point Mutation; Fluorescent Dyes; Hemolysin Proteins; Cysteine