Vertical collapse of a cytolysin prepore moves its transmembrane β-hairpins to the membrane

Abstract

Perfringolysin O (PFO) is a prototype of the large family of pore-forming cholesterol-dependent cytolysins (CDCs). A central enigma of the cytolytic mechanism of the CDCs is that their membrane-spanning b-hairpins (the transmembrane amphipathic b-hairpins (TMHs)) appear to be B40 Å too far above the membrane surface to cross the bilayer and form the pore. We now present evidence, using atomic force microscopy (AFM), of a significant difference in the height by which the prepore and pore protrude from the membrane surface: 11375 Å for the prepore but only 7375 Å for the pore. Time-lapse AFM micrographs show this change in height in real time. Moreover, the monomers in both complexes exhibit nearly identical surface features and these results in combination with those of spectrofluorimetric analyses indicate that the monomers remain in a perpendicular orientation to the bilayer plane during this transition. Therefore, the PFO undergoes a vertical collapse that brings its TMHs to the membrane surface so that they can extend across the bilayer to form the b-barrel pore.

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

“…1 A), and major conformational changes would be necessary to bring the TMHs in D3 to the bilayer surface for insertion. Such changes are compatible with atomic force microscopy (AFM) data that show a large difference in the height above the membrane surface of the prepore complex (113 Ϯ 5 Å) and the inserted pore complex (73 Ϯ 5 Å) (13). This study also indicated, consistent with earlier work (12), that the polypeptide segments destined to form the TMHs do not contact the bilayer at the prepore stage of pore formation.…”

“…However, the FRET measurements agree very well with the heights of the prepore and pore complexes determined by using AFM. Czajkowsky et al (13) reported a distance of 73 Ϯ 5 Å between the top of the PFO pore complex and the membrane surface, which is very similar to the 65 Å distance of closest approach between the D1 fluorophore and the As at the membrane surface ( Table 1). The observed changes in the height of the PFO complex during the prepore-to-pore transition were also similar: Ϸ40 Å by AFM (13) and Ϸ34 Å by FRET (Table 1).…”

“…Czajkowsky et al (13) reported a distance of 73 Ϯ 5 Å between the top of the PFO pore complex and the membrane surface, which is very similar to the 65 Å distance of closest approach between the D1 fluorophore and the As at the membrane surface ( Table 1). The observed changes in the height of the PFO complex during the prepore-to-pore transition were also similar: Ϸ40 Å by AFM (13) and Ϸ34 Å by FRET (Table 1). Thus, two independent approaches agree on the magnitude of the structural change in the overall height of the PFO complex during pore formation.…”

“…Data from collisional quenching experiments indicate that the TMH residues do not come into contact with the hydrophobic core of the bilayer even at the prepore stage of pore formation (12,13). However, very little is known about the positioning of these TMH segments relative to the membrane surface before their insertion into the bilayer.…”

What do diffusion measurements tell us about membrane compartmentalisation? Emergence of the role of interprotein interactions

“…For example, AFM has provided an understanding of oligomeric Rho protein structure in native membranes [78]. Recent progress in time-lapse AFM showed, in real time, the change in height of the pore protrusion from the membrane of cytolysin within its formation [79]. Scheuring and Sturgis [80] have even managed to image photosynthetic membranes with a spatial resolution of 1nm.…”