Staphylococcal alpha-toxin: the role of the N-terminus in formation of the heptameric pore -- a fluorescence study.
Staphylococcus aureus alpha-toxin forms heptameric pores on eukaryotic cell membranes. Assembly of the heptamer precedes formation of the transmembrane pore. The latter event depends on a conformational change that drives a centrally located stretch of 15 amino acid residues into the lipid bilayer. A second region of the molecule that has been implicated in the pre-pore to pore transition is the far N-terminus. Here, we used fluorescently labeled single cysteine replacement mutants to analyze the functional role of the far N-terminus of alpha-toxin. Pyrene attached to mutants S3C, I5C and 17C forms excimers within the toxin pore complex. This indicates that the distance of adjacent N-termini is less than 10-12 Angstrom. By labeling with the polarity-sensitive fluorophore acrylodan, pore formation is shown to cause distinct environmental changes in the N-terminus. Removal of membrane lipids from the labeled heptamers has no effect upon the acrylodan spectrum, indicating lack of direct contact of the N-terminus with the target membrane. The environmental alterations to the N-terminus are thus due to altered protein structure only. Both acrylodan emission shifts and pyrene excimers were shown to be absent in toxin heptamers that were arrested at the pre-pore stage. Therefore, while not being directly involved in membrane penetration, the N-termini of the alpha-toxin heptamer subunits move into immediate mutual proximity concomitantly with transmembrane pore formation.
Valeva
A
A
Institute of Medical Microbiology, University of Mainz, Germany.
Pongs
J
J
Bhakdi
S
S
Palmer
M
M
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry*; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D015394:Molecular Structure; D011487:Protein Conformation; D011721:Pyrenes; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus
D001427:Bacterial Toxins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D011721:Pyrenes; C021091:staphylococcal alpha-toxin; C038518:acrylodan; C030984:pyrene; D015081:2-Naphthylamine