SUBUNIT STOICHIOMETRY OF STAPHYLOCOCCAL ALPHA-HEMOLYSIN IN CRYSTALS AND ON MEMBRANES - A HEPTAMERIC TRANSMEMBRANE PORE

Elucidation of the accurate subunit stoichiometry of oligomeric membra ne proteins is fraught with complexities. The interpretations of chemi cal cross-linking, analytical ultracentrifugation, gel filtration, and low-resolution electron microscopy studies are often ambiguous. Staph ylococcal alpha-hemolysin (alpha HL), a homooligomeric toxin that form s channels in cell membranes, was believed to possess six subunits arr anged around a sixfold axis of symmetry. Here, we report that analysis of x-ray diffraction data and chemical modification experiments indic ate that the alpha HL oligomer is a heptamer. Self-rotation functions calculated using x-ray diffraction data from single crystals of alpha HL oligomers show a sevenfold axis of rotational symmetry. The alpha H L pore formed on rabbit erythrocyte membranes was determined to be a h eptamer by electrophoretic separation of alpha HL heteromers formed fr om subunits with the charge of wild-type alpha HL and subunits with ad ditional negative charge generated by targeted chemical modification o f a single-cysteine mutant. These data establish the heptameric oligom erization state of the alpha HL transmembrane pore both in three-dimen sional crystals and on a biological membrane.