Abstract
Elucidation of the accurate subunit stoichiometry of oligomeric membrane proteins is fraught with complexities. The interpretations of chemical cross-linking, analytical ultracentrifugation, gel filtration, and low-resolution electron microscopy studies are often ambiguous. Staphylococcal alpha-hemolysin (alpha HL), a homooligomeric toxin that forms channels in cell membranes, was believed to possess six subunits arranged around a sixfold axis of symmetry. Here, we report that analysis of x-ray diffraction data and chemical modification experiments indicate 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 HL pore formed on rabbit erythrocyte membranes was determined to be a heptamer by electrophoretic separation of alpha HL heteromers formed from subunits with the charge of wild-type alpha HL and subunits with additional negative charge generated by targeted chemical modification of a single-cysteine mutant. These data establish the heptameric oligomerization state of the alpha HL transmembrane pore both in three-dimensional crystals and on a biological membrane.