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.
Lesieur
C
C
Department of Biochemistry, University of Geneva, 30 quai E. Ansermet, 1211 Genève 4, Switzerland.
Frutiger
S
S
Hughes
G
G
Kellner
R
R
Pattus
F
F
van der Goot
F G
FG
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D008969:Molecular Sequence Data; D009154:Mutation; D052899:Pore Forming Cytotoxic Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D014508:Urea
D001427:Bacterial Toxins; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin; D014508:Urea