Increased stability upon heptamerization of the pore-forming toxin aerolysin.

Aerolysin is a bacterial pore-forming toxin that is secreted as an inactive precursor, which is then processed at its COOH terminus and finally forms a circular heptameric ring which inserts into membranes to form a pore. We have analyzed the stability of the precursor proaerolysin and the heptameric complex. Equilibrium unfolding induced by urea and guanidinium hydrochloride was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to the unfolding of the large COOH-terminal lobe followed by the unfolding of the small NH(2)-terminal domain. We show that proaerolysin contains two disulfide bridges which strongly contribute to the stability of the toxin and protect it from proteolytic attack. The stability of aerolysin was greatly enhanced by polymerization into a heptamer. Two regions of the protein, corresponding to amino acids 180-307 and 401-427, were identified, by limited proteolysis, NH(2)-terminal sequencing and matrix-assisted laser desorption ionization-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer interactions in the heptameric protein and are exclusively composed of beta structure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenerative diseases.

Membrane insertion: The strategies of toxins (review).

Protein toxins are soluble molecules secreted by pathogenic bacteria which act at the plasma membrane or in the cytoplasm of target cells. They must therefore interact with a membrane at some point, either to modify its permeability properties or to reach the cytoplasm. As a consequence, toxins have the built-in capacity to adopt two generally incompatible states: water-soluble and transmembrane. Irrespective of their origin or function, the membrane interacting domain of most protein toxins seems to have adopted one out of two structural strategies to be able to undergo this metamorphosis. In the first group of toxins the membrane interacting domain has the structural characteristics of most known membrane proteins, i.e. it contains hydrophobic and amphipathic alpha-helices long enough to span a membrane. To render this 'membrane protein' water-soluble during the initial part of its life the hydrophobic helices are sheltered from the solvent by a barrel of amphipathic helices. In the second group of toxins the opposite strategy is adopted. The toxin is an intrinsically soluble protein and is composed mainly of beta-structure. These toxins manage to become membrane proteins by oligomerizing in order to combine amphipathic beta-sheet to generate sufficient hydrophobicity for membrane insertion to occur. Toxins from this latter group are thought to perforate the lipid bilayer as a beta-barrel such as has been described for bacterial porins, and has recently been shown for staphylococcal alpha-toxin. The two groups of toxins will be described in detail through the presentation of examples. Particular attention will be given to the beta-structure toxins, since four new structures have been solved over the past year: the staphyloccocal alpha-toxin channel, the anthrax protective antigen protoxin, the anthrax protective antigen-soluble heptamer and the CytB protoxin. Structural similarities with mammalian proteins implicated in the immune response and apoptosis will be discussed. Peptide toxins will not be covered in this review.

Conformational changes due to membrane binding and channel formation by staphylococcal alpha-toxin.

Conformational changes occurring upon membrane binding and subsequent insertion of staphylococcal alpha-toxin were studied using complementary spectroscopic techniques. Experimental conditions were established where binding could be uncoupled from membrane insertion but insertion and channel formation seemed to be concomitant. Binding led to changes in tertiary structure as witnessed by an increase in tryptophan fluorescence, a red shift of the tryptophan maximum emission wavelength, and a change in the near UV CD spectrum. In contrast to what was observed for the soluble form of the toxin, 78% of the tryptophan residues in the membrane-bound form were accessible to the hydrophilic quencher KI. At this stage, the tryptophan residues were not in the immediate vicinity of the lipid bilayer. Upon membrane insertion, a second conformational change occurred resulting in a dramatic drop of the near UV CD signal but an increase of the far UV signal. Tryptophan residues were no longer accessible to KI but could be quenched by brominated lipids. In the light of the available data on channel formation by alpha-toxin, our results suggest that the tryptophan residues might be dipping into the membrane in order to anchor the extramembranous part of the channel to the lipid bilayer.

Protonation of histidine-132 promotes oligomerization of the channel-forming toxin aerolysin.

Aerolysin is a bacterial toxin that binds to a receptor on eukaryotic cells and oligomerizes to form stable, SDS-resistant, noncovalent oligomers that insert into the plasma membrane and produce well-defined channels. Little is known about the mechanisms controlling this process. Here we show that the protonation of a single histidine is required for oligomerization of aerolysin in solution. First we have investigated the effect of pH on the activity of aerolysin. The toxin's ability to disrupt human erythrocytes declined as the pH increased above 7.4. Experiments with receptor-free planar lipid bilayers demonstrated that the rate at which aerolysin formed channels also decreased with increasing pH, although the conductance of preexisting channels was not affected. The reduction in the rate of channel formation was shown to be due to a decrease in the toxin's ability to oligomerize. Our data indicate that the pH effect on activity is due to the deprotonation of a single residue rather than a global effect of pH on the protein. In agreement with our previous site-directed mutagenesis studies, His-132 is most likely to be the target of this pH effect. This conclusion was reinforced by the fact that we could shift the pH dependence of the activity to lower pH values by mutating Asp-139, a residue less than 3 A away from His-132 and likely to contribute to the usually high pKa of this histidine.