Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin.

Abstract

Staphylococcus aureus alpha‐toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores. During assembly, the formation of a pre‐pore precedes membrane permeabilization; the latter is linked to a conformational change in the oligomer. Here, 41 single‐cysteine replacement toxin mutants were thiol‐specifically labelled with the polarity‐sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attached to residues in the sequence 118–140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluorophore to a hydrophobic environment. Within this region, two functionally distinct parts could be identified. For mutants at positions 126–140, the shifts were partially reversed after membrane solubilization by detergents, indicating a direct interaction of the label with the membrane lipids. Membrane insertion of this sequence occurred together with the final pre‐pore to pore transition of the heptamer. Thus residues 126–140 constitute a transmembrane sequence in the pore. With labelled residues 118–124, pre‐pore assembly was the critical event to induce the spectral shifts, which persisted after the removal of membrane lipids and hence probably reflects protomer‐protomer contacts within the heptamer. Finally, a derivative of the mutant N121C yielded occluded pores which could be opened by reductive reversal of the modification. Therefore this residue probably lines the lumen of the pore.

Elimination of a bacterial pore‐forming toxin by sequential endocytosis and exocytosis

“…Wild type S. aureus a-toxin and single amino acid exchange mutant D152C were bacterially expressed and purified to homogeneity as described [16]. Internally radiolabeled a-toxin of high specific activity ($3000 GBq/mmol) was generated by coupled in vitro transcription/translation [32].…”

“…Labeled non-lytic D152C, and fully active single amino acid exchange mutant K21C (referred to as wild type toxin), were thereby obtained. Hemolytic activity was measured as described [16].…”

“…It is secreted as a monomer of 34 kDa and binds to a wide variety of target cells. Membrane-associated a-toxin monomers assemble into heptameric ring-like structures, which can insert an amphipathic amino acid sequence to generate a b-barrel traversing the lipid bilayer [15,16]. In erythrocytes, a-toxin induces colloidosmotic lysis [17], whereas attack on nucleated target cells causes necrosis [18] or apoptosis [19].…”

Probing single nanometer-scale pores with polymeric molecular rulers

“…For example, scanning cysteine mutagenesis and covalent chemical modification were used to identify amino acid side chains that line the pore of the nicotinic acetylcholine receptor channel, 5 the Staphylococcus aureus ␣-hemolysin ͑␣HL͒ channel, [6][7][8][9][10][11][12][13][14] and the active form of Bacillus anthracis protective antigen ͑i.e., PA 63 ͒. 15,16 Nonelectrolyte water soluble polymers have been used to deduce channel structural features.…”

Platelet-Bacterial Interactions as Therapeutic Targets in Infective Endocarditis

“…Its expression is accessory-gene regulon (agr)-regulated and is secreted into the extracellular environment as a monomeric water soluble protein (Ikigai and Nakae, 1985). The toxin disrupts the cell membranes by binding to the lipid bilayer, forming an oligomeric structure that forms a water filled transmembrane pore (Valeva et al, 1996). Studies have demonstrated that the toxin has primarily two modes of interaction with host cells.…”