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

Abstract

α-Toxin is secreted by Staphylococcus aureus as a soluble monomer that without further activation can oligomerize and form a transmembrane channel. One of the aims of the present work was to understand the underlying mechanisms of the membrane insertion process. We first showed that α-toxin undergoes a native to molten globule transition at acidic pH. We then demonstrated that the variation in kinetics of channel formation in negatively charged lipid vesicles increased as a function of the interfacial pH correlated with the appearance of the acidic molten globule state. Further studies indicated that unfolding occurred in several steps presumably reflecting the existence of independent folding units. The different unfolding steps could be selectively affected by varying the temperature or the ionic strength. However, unfolding was only partial as the secondary structure remained native-like as shown by far UV CD spectroscopy. Membrane insertion correlated with the first unfolding step.

We then analysed the different steps leading to channel formation. Membrane binding and oligomerization could never be separated under our experimental conditions suggesting they were concomitant. This step was accompanied by a significant conformational change as indicated by the fact that the tryptophan residues, which were shielded in the soluble monomer, became accessible to the hydrophilic quencher KI. Moreover the membrane bound pre-pore had higher thermal stability than the soluble monomer and was protease resistant. Upon subsequent membrane insertion, a second conformational change occurred during which the tryptophan residues became buried as indicated by the fact that they could be quenched by the brominated lipids. This change in environment of 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 observation that the transmembrane channel was protease sensitive and did not undergo any thermal transition upon heating.

The present work indicate that the α-toxin monomer has to undergo a transition to a molten globule state in order to become competent for membrane insertion. Moreover some of the characteristics of this highly flexible intermediate state are retained in the final channel form of the toxin as illustrated by the fact that the molten globule in solution and the transmembrane channel are sensitive to pronase at the same sites. The above observation shed new light on the structure of the α-toxin channel. Indeed it was thought to be a rigid structure. In contrast our data show that α-toxin forms a rather flexible complex in the membrane. This flexibility might be important for the channel-gating mechanism and demonstrating this will be a challenge for the future.