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 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.
Valeva
A
A
Institute of Medical Microbiology and Hygiene, University of Mainz, Germany.
Weisser
A
A
Walker
B
B
Kehoe
M
M
Bayley
H
H
Bhakdi
S
S
Palmer
M
M
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D008081:Liposomes; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus
D001427:Bacterial Toxins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D008081:Liposomes; C021091:staphylococcal alpha-toxin; C038518:acrylodan; D015081:2-Naphthylamine; D003545:Cysteine