title: Assembly of the oligomeric membrane pore formed by Staphylococcal alpha-hemolysin examined by truncation mutagenesis.
abstract: The alpha-hemolysin (alpha HL) from Staphylococcus aureus causes the lysis of susceptible cells such as rabbit erythrocytes (rRBCs). Lysis is associated with the formation of a hexameric pore in the plasma membrane. Here we show that truncation mutants of alpha HL missing 2 to 22 N-terminal amino acids form oligomers on the surfaces of rRBCs but fail to lyse the cells. By contrast, mutants missing 3 or 5 amino acids at the C terminus are very inefficient at oligomerization but do lyse rRBCs, albeit extremely slowly. The C-terminal truncation mutants, retarded as monomers on the cell surface, undergo a conformational change in which the protease-sensitive loop located near the midpoint of the polypeptide chain becomes occluded. Judged by this criterion, polypeptides truncated at the N terminus, frozen as nonlytic oligomers, are in a similar conformation. A second proteolytic site near the N terminus of the polypeptide becomes inaccessible in the lytic pore formed by the wild-type polypeptide, supporting the idea that a second conformational change occurs upon pore formation. These findings suggest a pathway for assembly of the lytic pore in which alpha HL first binds to target cells as a monomer, which is converted to a nonlytic oligomeric intermediate before formation of the pore. In keeping with this model, an N-terminal truncation mutant blocks the slow lysis induced by a C-terminal truncation mutant, presumably by diverting the weakly lytic subunits into inactive oligomers.
authors.affiliation: Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
authors.forename: B
authors.initials: B
authors.lastname: Walker
authors.forename: M
authors.initials: M
authors.lastname: Krishnasastry
authors.forename: L
authors.initials: L
authors.lastname: Zorn
authors.forename: H
authors.initials: H
authors.lastname: Bayley
mesh_terms: D000595:Amino Acid Sequence; D001345:Autoradiography; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D013210:Staphylococcus
chemical_list: D001427:Bacterial Toxins; D004277:DNA, Single-Stranded; D006460:Hemolysin Proteins; D008565:Membrane Proteins; C021091:staphylococcal alpha-toxin