Membrane Insertion of the Heptameric Staphylococcal α-Toxin Pore

Abstract

Staphylococcal ␣-toxin forms heptameric pores on eukaryotic cells. After binding to the cell membrane in its monomeric form, the toxin first assembles into a heptameric pre-pore. Subsequently, the pre-pore transforms into the final pore by membrane insertion of an amphipathic ␤-barrel, which comprises the "central loop" domains of all heptamer subunits. The process of membrane insertion was analyzed here using a set of functionally altered toxin mutants. The results show that insertion may be initiated within an individual protomer when its NH 2 terminus activates its central loop. The activated state is then shared with the central loops of the residual heptamer subunits, which results in cooperative membrane penetration. This cooperation of the central loops commences while these are still remote from the lipid bilayer. Nevertheless, it is subject to modulation by the target membrane, which therefore acts across a distance much like an allosteric effector. However, while allosteric transitions usually are reversible, membrane insertion of ␣-toxin is an irreversible event, and we show here that it can proceed to completion in a domino-like fashion when triggered by as little as a single foreign atom within the entire heptamer.

Role of the Amino Latch of Staphylococcal α-Hemolysin in Pore Formation

“…Therefore, the stimulation of the prepore to pore transition by the N terminus of ␣HL must be independent of the final conformation of the N terminus and its sequence. In an earlier study, Valeva and coworkers (52) provided evidence that, during heteroheptamer formation by certain pairs of mutant ␣HL subunits, the N terminus in one class of subunits can activate its own pre-stem intramolecularly and that this activation is transmitted intermolecularly within the prepore to the pre-stem of a normally defective subunit. Further, when the part of the pre-stem that becomes the lower half of the ␤ barrel is deleted, the resulting mutant assembles spontaneously in the absence of membranes to form heptameric structures in which the N termini have formed latches (37), again indicating a cooperative interaction between the N terminus and the pre-stem.…”

The Prediction of Amphiphilic α-Helices

“…Amphiphilic structures play a role in the biological interactions of many proteins and peptides that participate in membrane dependent processes. These membrane interactive structures may be classified as possessing either: primary, secondary or tertiary amphiphilicity, with each having a range of effects on lipid organisation [1,[12][13][14][15][16][17][18][19][20].…”

“…This arrangement of amino acid residues allows these structures to form a barrier between aqueous and hydrophobic environments and such structures are now recognised as major protein secondary structural elements, having an important role in a range of membrane dependent processes [1,[12][13][14][15][16][17][18][19][20]. Amphiphilic β-sheet occurs in a number of membrane interactive molecules including: the proteins translocation machinery of mitochondrial and plastid outer membranes [32], bacterial protein toxins [20] such as the heptameric β-barrel pore formed by molecules of staphylococcal α-toxin [13,[33][34][35] and bacterial porins [36]. Amphiphilic α-helices were recognised within the molecules of myoglobin and haemoglobin during the mid sixties [37] and in a major study, were later identified within apolipoproteins by Segrest et al, [38].…”

β-Barrel Pore-Forming Toxins: Intriguing Dimorphic Proteins

“…Biochemical analyses of R-HL pore formation have revealed two intermediate states: the membrane-bound monomer and the heptameric pre-pore complex (e.g., refs 39, 40, and 53). Oligomerization precedes membrane insertion during pore formation, and TMH insertion involves interaction among neighboring protomers (28,40,54,55). A comparison of the monomeric structure of LukF and the oligomeric structure of the homologous R-HL revealed that the β-sandwich and rim domains behave as rigid bodies that adopt different relative conformations in the monomer and the heptamer due to small changes spread over a number of residues at the junction between the β-sandwich and rim domains (Figure 1).…”