Disulfide-bond scanning reveals assembly state and beta-strand tilt angle of the PFO beta-barrel

Perfringolysin O (PFO), a bacterial cholesterol-dependent cytolysin, binds to a mammalian cell membrane, oligomerizes into a circular prepore complex (PPC), and forms a 250-Å transmembrane β-barrel pore in the cell membrane. Each PFO monomer has two sets of 3 short α-helices that unfold and ultimately refold into two transmembrane β-hairpin (TMH) components of the membrane-embedded β-barrel. Inter-strand disulfide bond scanning revealed that β-strands in a fully assembled PFOβ-barrel were strictly aligned and tilted at 20 ° to the membrane perpendicular. In contrast, in a low temperature-trapped PPC intermediate, the TMHs were unfolded and had sufficient freedom of motion to interact transiently with each other; yet the TMHs were not aligned or stably hydrogen-bonded. The PFO PPC-to-pore transition therefore converts TMHs in a dynamic folding intermediate far above the membrane into transmembrane β-hairpins that are hydrogen bonded to those of adjacent subunits in the bilayer-embedded β-barrel. Cholesterol-dependent cytolysins (CDCs) are a large family of secreted bacterial pore-forming toxins that specifically bind to cholesterol-containing mammalian membranes1. While the pore size (250–300 Å) and number of monomers per pore (35–50) can var