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 heptameri c complex. Equilibrium unfolding induced by urea and guanidinium hydrochlor ide was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to th e unfolding of the large COOH-terminal lobe followed by the unfolding of th e small NH2-terminal domain. We show that proaerolysin contains two disulfi de bridges which strongly contribute to the stability of the toxin and prot ect it from proteolytic attack. The stability of aerolysin was greatly enha nced by polymerization into a heptamer. Two regions of the protein, corresp onding to amino acids 130-307 and 401-427, were identified, by limited prot eolysis, NH2-terminal sequencing and matrix-assisted laser desorption ioniz ation-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer int eractions in the heptameric protein and are exclusively composed of beta st ructure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenera tive diseases.