Je. Gouaux et al., SUBUNIT STOICHIOMETRY OF STAPHYLOCOCCAL ALPHA-HEMOLYSIN IN CRYSTALS AND ON MEMBRANES - A HEPTAMERIC TRANSMEMBRANE PORE, Proceedings of the National Academy of Sciences of the United Statesof America, 91(26), 1994, pp. 12828-12831
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.