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...

Staphylococcal alpha-hemolysin is a water soluble, monomeric, bacterial exotoxin, which forms heptameric pores in membranes. The rate determining step in assembly is the conversion of a heptameric prepore to the fully assembled pore in which the central glycine-rich domain of each subunit inserts into the membrane to form a 14 strand beta barrel. Barrel formation is accompanied by a conformational change in which each N terminus latches onto an adjacent subunit. In the monomer in solution, the central domain is loosely organized and exposed to solvent. In this study, 25 amino acids of the central domain were removed and replaced with the sequence Asp-Gly, which favors the formation of a type I' beta-turn, to yield a mutant devoid of hemolytic activity. Within minutes after synthesis in the absence of membranes, the mutant polypeptide spontaneously assembled into heptamers, as demonstrated by atomic force microscopy. Limited proteolysis suggested that the N termini of the subunits in the heptamers were in the fully assembled pore conformation rather than the prepore conformation. Based on these findings, the deletion is proposed to constrain the central domain and thereby force the creation of a shortened beta barrel, which in turn induces the additional structural changes that normally accompany pore formation. The truncated pore might make a useful framework for the construction of designed membrane active macromolecules.

a-Hemolysin from Staphylococcus aureus assembles from a water-soluble, monomeric species to a membrane-bound heptamer on the surface of target cells, creating water-filled channels that lead to cell death and lysis. Staphylococcus aureus also produces the y-hemolysin and leukocidin toxins, which function as two component toxins in the disruption and lysis of erythrocytes and leukocytes. Analysis of the aligned sequences of a-hemolysin, y-hemolysin, and leukocidin in the context of the a-hemolysin heptamer structure supports the conclusion that even though the level of sequence identity between a-hemolysin and the y-hemolysin and leukocidin toxins is in the so-called twilight zone, the threedimensional structures of the protomers are probably conserved. By analogy with a-hemolysin, y-hemolysin and leukocidin may also form oligomeric, transmembrane channels in which an antiparallel /?-barrel constitutes the primary membrane-embedded domain.Keywords: a-hemolysin; y-hemolysin; heptameric channels; leukocidin; pore-forming bacterial toxins; Staphylococcus aureus; transmembrane /?-barrel Staphylococcus aureus, one of the most common human pathogens, secretes an arsenal of toxins that include a-hemolysin (aHL; Comparison of the sequences corresponding to the mature aHL, yHL and Luk polypeptides shows that the pairwise sequence identity between aHL and the other toxins ranges from 23% to 32%, using the FASTA alignment program (GCG, 1996). As described previously (Cooney et al., 1993), yHL is more closely related to Luk by primary sequence than aHL is related to yHL and Luk.Indeed, the level of sequence conservation between aHL and the yHL and Luk proteins borders on the so-called twilight zone, indicating that conservation of three-dimensional structure is not unambiguous. Multiple-sequence alignments of aHL and nine representative sequences of y H L and Luk using Clustal W (version 1.6; Thompson et al., 1994), MAP (Huang, 1994), and PIMA (Smith & Smith, 1990, operating from the web site http: //dot.imgen.bcm.tmc.edu:9331/multi-align at Baylor College of Medicine, gave similar results, although there were some specific differences between the alignments (Fig. 2). Relative to the aHL sequence, the sites of variation in the multiple sequence alignments using the three programs were located (1) at the amino terminus;

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aq-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate lowspin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64VIV68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy.In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixedvalence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm. Taken together these data suggest that the observation of Soret splitting, including a feature at 450 nm, results from specific protein-cofactor interactions that are unique to the cytochrome a-binding pocket of the terminal oxidases. The conservation of this unique binding pocket among evolutionarily distant species may reflect some mechanistic significance for this structure.

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