The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation

Abstract

FRET measurements were used to determine the domain-specific topography of perfringolysin O, a pore-forming toxin, on a membrane surface at different stages of pore formation. The data reveal that the elongated toxin monomer binds stably to the membrane in an "end-on" orientation, with its long axis approximately perpendicular to the plane of the membrane bilayer. This orientation is largely retained even after monomer association to form an oligomeric prepore complex. The domain 3 (D3) polypeptide segments that ultimately form transmembrane beta-hairpins remain far above the membrane surface in both the membrane-bound monomer and prepore oligomer. Upon pore formation, these segments enter the bilayer, whereas D1 moves to a position that is substantially closer to the membrane. Therefore, the extended D2 beta-structure that connects D1 to membrane-bound D4 appears to bend or otherwise reconfigure during the prepore-to-pore transition of the perfringolysin O oligomer.

Fig. 1.

PFO domains and possible rearrangements. (A) Ribbon representation of the crystal structure of monomeric water-soluble PFO (5) shows locations of E167 in D1 and A215 in D3 that were substituted with Cys and labeled with BODIPY. A cross-bar indicates locations of the two residues that formed an intramolecular disulfide bond after substitution by Cys; a star indicates the location of the F318A mutation. The image was generated by using molscript. (B) Cartoons of potential domain rearrangements as PFO first binds to the membrane and then oligomerizes before forming the inserted pore complex.

Fig. 2.

Spectral overlap. The corrected emission spectrum of 6 nM rPFO^DS(A215C-BODIPY) (solid line) and the absorbance spectrum of Rh-PE (2 mol percentage) (dotted line) in cholesterol-containing vesicles (0.5 mM total lipid) are shown.

Fig. 3.

Dependence of energy transfer upon acceptor density. Q_D/Q_DA values for BODIPY-labeled D1 (A) or D3 (B) at different stages of pore formation and at three different acceptor densities. Best-fit lines were required to go through 0, 1. No line is shown for the pore complex data in B because L ≪ R₀ and Eq. 1 does not apply.

Fig. 4.

FRET-detected changes in PFO topography. Based on domain-specific FRET measurements, the orientation of membrane-bound PFO at different stages before membrane insertion is shown and described in the text. For simplicity, only one monomer in the prepore (iii) and pore (iv) complexes is shown.