1.C.3 The α-Hemolysin Channel-forming Toxin (αHL) Family
The α-hemolysin (αHL; α-toxin, alpha-toxin) of the human pathogen Staphylococcus aureus is secreted as a 33 kDa monomer. This monomeric species associates with animal cell membranes to form a 232 kDa homoheptameric transmembrane β-barrel pore that promotes cell death by allowing bilayer permeability to ions, water and small solutes, thereby promoting cell lysis. The three-dimensional structure of αHL has been solved by x-ray crystallography to 1.9 Å resolution (Song et al., 1996). αHL forms a solvent-filled channel with a length of 100 Å, that runs along the seven-fold axis of the protein and ranges from 14 to 46 Å in diameter. The transmembrane domain of the mushroom-shaped heptamer is the lower portion of the mushroom, consisting of a 14-strand antiparallel β-barrel to which each protomer contributes two α-strands, each 65 Å long. The interior of the β-barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 Å wide. The pore can transport peptides, and charged residues in the pore influences the rate of passage (Wolfe et al., 2007; Hammerstein et al., 2011). αHL has been used to create a system with directional control of a processive molecular hopper (Qing et al. 2018).
Several Staphylococcal toxins of this family are two-component cytolysins. These toxins include α-hemolysin (Hlg: Hlg1 (LukF) Hlg2), leukocidin (Luk: LukF LukS) and Pantone-Valentine leukocidin (Luk-PV: LukF-PV LukS-PV). They have 7 subunits arranged in a ring with alternate subunit arrangements (subunit stoichiometries of 3:4 or 4:3 (Sugawara-Tomita et al., 2002). Each toxin has specificity for different mammalian cell types and hosts. LukF (also called Hlg1) and LukF-PV comprise class F while Hlg2, LukS and LukS-PV comprise class S. Proteins of each class are 70% identical, but the proteins are about 30% identical between classes. They are 20-30% identical to the single component Staphylococcal α-hemolysin described above. Because the two components (LukS and LukF) comprise two distinct subfamilies of the αHL family, they are listed under two different TC numbers (#1.C.3.3.1 and #1.C.3.4.1).
The αHL family consists of pore-forming toxins from Staphylococcal species, Bacillus cereus, B. anthracis and Clostridium perfringens. They are distantly related to the CHL family of pentameric toxins from Gram-negative bacteria (TC #1.C.14). The S. aureus protein monomers are 308-326 residues in length, while the B. cereus protein is of 412 residues and the C. perfringens monomers is of 336 residues. α-HL forms two subpopulations of ion conducting channels. Furini et al. (2008) provided evidence that this toxin can form both hexameric and heptameric pores, accounting for the ion conuctance results.
The phylogenetic tree for the αHL family reveals four clusters (Saier et al., 1999). The Staphylococcus α-hemolysin for which the three-dimensional structure is available comprises one branch, the B. cereus and C. perfringens proteins comprise a second, and all other members of the family fall into the remaining two clusters.
α-haemotoxins are members of the αHL family that form heterooligomeric (bicomponent) toxins (HlgA · HlgB or HlgB · HlgC). Both form pores in lipid membranes with conductances, current-voltage characeristics and stability properties similar to α-toxin. However, they are cation selective rather than anion selective. There is a conserved region at the pore entrance with four basic residues in α-toxin but either basic or acidic residues in α-haemolysins. These residues form an entrance electrostatic filter (Comai et al. 2002).
Cracknell et al. (2013) described the translocation of ssRNA heteropolymers (91-6083 bases) through the α-hemolysin nanopore. Translocation of these long ssRNAs is characterized by surprisingly long, almost complete ionic current blockades with durations averaging milliseconds per base (at +180 mV). The event durations decrease exponentially with increased transmembrane potential but are largely unaffected by the presence of urea. When the ssRNA is coupled at the 3' end to streptavidin, which cannot translocate through the pore, permanent blockades are observed, supporting the conclusion that the transient blockage of current arises from ssRNA translocation. Asandei et al. 2016 have described the dynamics of a single peptide as it passes across a voltage-biased alpha-hemolysin nanopore.
PLEKHA7 and other junctional proteins are host factors mediating death by S. aureus alpha-toxin. ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C-terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. Thus, a dock-and-lock molecular mechanism targets ADAM10 to junctions, providing a paradigm for how junctions may regulate transmembrane receptors through their clustering (Shah et al. 2018).
The generalized transport reaction catalyzed by these pore-forming toxins is:
Small molecules (in) Small molecules (out)