Conformational changes in Staphylococcus aureus' alpha-toxin : from water-soluble monomer to a transmembrane channel

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.