Abstract
Alpha-toxin is secreted by Staphylococcus aureus as a soluble monomer
that withoutfurther activation can oligomerize and form a transmembrane
channel. One of the aimsof the present work was to understand the
underlying mechanisms of the membrane insertionprocess. We first showed
that alpha toxin undergoes a native to molten globule transitionat acidic
pH. We then demonstrated that the variation in kinetics of channel
formationin negatively charged lipid vesicles increased as a function of
the interfacial pHcorrelated with the appearance of the acidic molten
globule state. Further studiesindicated that unfolding occurred in
several steps presumably reflecting the existenceof independent folding
units. The different unfolding steps could be selectivelyaffected by
varying the temperature or the ionic strength. However, unfolding wasonly
partial as the secondary structure remained native-like as shown by far
UV CDspectroscopy. Membrane insertion correlated with the first unfolding
step.
We then analysed the different steps leading to channel formation.
Membrane bindingand oligomerization could never be separated under our
experimental conditions suggestingthey were concomitant. This step was
accompanied by a significant conformationalchange as indicated by the
fact that the tryptophan residues, which were shieldedin the soluble
monomer, became accessible to the hydrophilic quencher KI. Moreoverthe
membrane bound pre-pore had higher thermal stability than the soluble
monomerand was protease resistant. Upon subsequent membrane insertion, a
second conformationalchange occurred during which the tryptophan residues
became buried as indicated bythe fact that they could be quenched by the
brominated lipids. This change in environmentof the aromatic residues was
confirmed by the fact that the near UV CD spectrum collapsed.This
increased flexibility of the complex was in turn confirmed by the
observationthat the transmembrane channel was protease sensitive and did
not undergo any thermaltransition upon heating.
The present work indicate that the alpha-toxin monomer has to undergo a
transitionto a molten globule state in order to become competent for
membrane insertion. Moreoversome of the characteristics of this highly
flexible intermediate state are retainedin the final channel form of the
toxin as illustrated by the fact that the moltenglobule in solution and
the transmembrane channel are sensitive to pronase at thesame sites. The
above observation shed new light on the structure of the alpha
toxinchannel. Indeed it was thought to be a rigid structure. In contrast
our data showthat alpha-toxin forms a rather flexible complex in the
membrane. This flexibilitymight be important for the channel-gating
mechanism and demonstrating this will bea challange for the future.
Key words: Staphylococcal alpha-toxin; membrane-protein interaction; pore
formingtoxins.
