Subunit stoichiometry of staphylococcal alpha-hemolysin in crystals and on membranes: a heptameric transmembrane pore.
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.
Gouaux
J E
JE
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637.
Braha
O
O
Hobaugh
M R
MR
Song
L
L
Cheley
S
S
Shustak
C
C
Bayley
H
H
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry*; D011817:Rabbits; D011994:Recombinant Proteins
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011994:Recombinant Proteins; C021091:staphylococcal alpha-toxin; D003545:Cysteine