Assembly of the oligomeric membrane pore formed by Staphylococcal alpha-hemolysin examined by truncation mutagenesis.

Journal article

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: Walker, B; Krishnasastry, M; Zorn, L; Bayley, J

• Publication status: Published
• Journal: Journal of biological chemistry
• Volume: 267
• Issue: 30
• Pages: 21782-21786
• Publication date: 1992-10-01
• EISSN: 1083-351X
• ISSN: 0021-9258
• Language: English
• Keywords: Mutagenesis; Membrane Proteins; Base Sequence; Hydrolysis; Staphylococcus; DNA, Single-Stranded; Hemolysin Proteins; Molecular Sequence Data; Protein Conformation; Amino Acid Sequence; Bacterial Toxins; Electrophoresis, Polyacrylamide Gel; Autoradiography