Subunit stoichiometry of staphylococcal alpha-hemolysin in crystals and on membranes: a heptameric transmembrane pore.

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 a-hemolysin (crHL), 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 aHlL oligomer is a heptamer. Self-rotation functions calculated using x-ray diffraction data from single crystals of acHL oligomers show a sevenfold axis of rotational symmetry. The aHiL pore formed on rabbit erythrocyte membranes was determined to be a heptamer by electrophoretic separation of aHL heteromers formed from subunits with the charge of wild-type aHL 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 aHlL transmembrane pore both in three-dimensional crystals and on a biological membrane.As exemplified by studies on cholera toxin (1), chemical cross-linking and electron microscopy may not provide precise and unequivocal information on the subunit stoichiometry of oligomeric proteins. Complete cross-linking of an oligomeric protein can give both cyclic and linear species, which may migrate differently on denaturing polyacrylamide gels, giving the false indication of an additional subunit (1). Dynamic or static rotational disorder about the axis of molecular symmetry in two-dimensional crystals can yield misleading diffraction data (1). Resolution of the subunit composition of cholera toxin was achieved by the collection and analysis of high-resolution x-ray diffraction data (2). A second example is aerolysin, a pore-forming protein secreted by Aeromonas hydrophila. Aerolysin was thought to form a pentamer or a hexamer (3) until a recent examination by rotational correlation analysis of particles in averaged electron micrographs suggested that it is a heptamer (4). However, this conclusion is being reevaluated (5). The gap junction connexon provides another example of an oligomeric membrane protein for which the number of subunits is still unclear. The connexon was believed to be a hexamer (6), but recent work suggests that it may be a pentamer (7).Reservations concerning the reliability of methods employed in determining the subunit stoichiometry of other membrane proteins have led us to reexamine the quaternary structure of a-hemolysin (aHL), a protein that is a model system for studying membrane protein assembly (8-11), and that has potential applications in biotechnology [e.g., as a component of immunotoxins (12) or an element in biosensors (13)]. The aHL polypeptide of 293 amino acids is secreted by Staphylococcus aureus as a water-soluble monomer and assembles upon contact with lipid bilayers or the deterg...

Glycerol Monolaurate Inhibits the Effects of Gram-Positive Select Agents on Eukaryotic Cells

“…Staphylococcal α-hemolysin forms heptamers in solution with the oligomeric forms embedding in the host red blood cell membrane to cause lysis (5). Our studies demonstrated that α-hemolysin was unable to bind to red blood cells in the presence of GML, suggesting that the primary effect of GML was to stabilize red blood cell membranes and block the binding of α-hemolysin to red blood cell membranes.…”

Characterisation of the heptameric pore‐forming complex of the Aeromonas toxin aerolysin using MALDI‐TOF mass spectrometry

“…This toxin, which has little sequence homology to aerolysin but shares a similar mode of action, was thought previously to form hexamers or tetramers based on electron microscopy [11], analytical centrifugation, cross-linking and SDS-PAGE analysis, [12,13]. However, X-ray analysis of crystals and an elegant charge shift migration assay have recently provided convincing evidence that alpha toxin forms a heptamer [32]. It has also been recently reported that the oligomeric forms of the protective antigen of anthrax toxin [33] as well as that of Vac A from Helicabacter pylori (R. Rappuoli, personal communication) are both heptamers.…”

The Role of the Amino Terminus in the Kinetics and Assembly of α-Hemolysin of Staphylococcus aureus

“…However, based on the electrophoretic migration, ␣-HL(3-293) was proposed to form a pentamer. Recently, the number of ␣-HL monomeric units present in the oligomer was revised from six to seven (23), and hence, the earlier conclusion that the ␣-HL(3-293) mutant forms a pentameric structure is no longer tenable. As discussed later, data presented here clearly show that the oligomer of ␣-HL(5-293) comigrates with the oligomer of ␣-HL (see Fig.…”