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

• Published in: The EMBO journal 1996-04, Vol.15 (8), p.1857-1864
• Main Authors: Valeva, A., Weisser, A., Walker, B., Kehoe, M., Bayley, H., Bhakdi, S., Palmer, M.
• Format: Article
• Language: English
• Subjects: 2-Naphthylamine - analogs & derivatives; Amino Acid Sequence; Bacterial Toxins - chemistry; Bacterial Toxins - genetics; Binding Sites; Cell Membrane - chemistry; Cysteine - chemistry; Fluorescent Dyes; Hemolysin Proteins - chemistry; Hemolysin Proteins - genetics; Liposomes; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Point Mutation; Protein Conformation; Spectrometry, Fluorescence; Staphylococcus aureus
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1996.tb00536.x

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.