Vertical collapse of a cytolysin prepore moves its transmembrane beta-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 beta-hairpins (the transmembrane amphipathic beta-hairpins (TMHs)) appear to be approximately 40 A 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: 113+/-5 A for the prepore but only 73+/-5 A 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 beta-barrel pore.

Figures

Figure 1

Structure of the water-soluble PFO monomer. A ribbon representation of the crystal structure of PFO is shown (Rossjohn et al, 1997). The α-helical bundles (TMH1 and TMH2) located in domain 3 (D3) that insert into the membrane in the pore complex are colored red and yellow. The six domain 3 α-helices unfurl into two amphipathic β-hairpins that insert into the membrane to form the transmembrane β-barrel (Shepard et al, 1998; Shatursky et al, 1999). Domain 4 (D4) has been shown to anchor PFO to the membrane in the prepore and pore complexes via the undecapeptide and three other short hydrophobic loops at its tip (Heuck et al, 2000; Ramachandran et al, 2002) (shown in yellow). This domain exists in a perpendicular orientation to the membrane and, except for its tip, is surrounded by the aqueous milieu (Ramachandran et al, 2002). Also shown in a space-filled mode are the positions of various residues studied herein (magenta) and the location of residues Y181 and the C190-C57 disulfide (white). The representations were generated using MOLMOL (Koradi et al, 1996).

Figure 2

AFM images of the PFO prepore complex associated with supported lipid bilayers containing cholesterol. (A) The prepore-trapped PFOY181C complexes form a largely uniform population of ring structures, 38 nm in outer diameter. Scale bar: 100 nm. (B) These complexes measure 113±5 Å high from the top of the membrane surface, similar to the height of the water-soluble monomer (Figure 1). The darkest region in this figure is a large defect in the membrane, where the tip is directly in touch with the mica substrate. Scale bar: 100 nm. (C) At smaller scan sizes, higher resolution features are clearly discerned, including the 25 Å periodic arrangement of subunits in the complex and a slight 7 Å decrease in height of each subunit from its outermost to its innermost edge. Scale bars: x, y, 25 nm; z, 10 nm.

Figure 3

AFM images of the PFO pore complexes in supported lipid bilayers that contain cholesterol. (A) Pore-forming PFO self-assembles into both ring and arc-shaped complexes similar in size to the prepore complexes. Scale bar: 100 nm. (B) The pore complexes protrude 73±5 Å from the bilayer, approximately 40 Å less than the prepore complexes. The darkest region on the left in this figure is a large defect in the membrane, where the tip is directly in touch with the mica substrate. Scale bar: 100 nm. (C) Smaller scan size images of these complexes reveal a number of finer features of the complexes, including a similar 25 Å periodicity of subunits in the complex and 7 Å decrease in the height of each subunit from its outer to inner edge. Comparison with the prepore complexes (Figure 2) shows that the surface contours of the protein in the prepore and pore complexes are the same. Scale bar: x, y, 25 nm; z, 10 nm.

Figure 4

Direct observation of the change in height upon the conversion of prepore complexes to pores. Sequential images (from left to right) of the same oligomeric complexes of PFOS190C-G57C obtained after the addition of DTT are shown. The first image (0 min) was obtained 30 min after the application of the reducing agent. The complexes are initially at a height of ∼113 Å and decrease to a height of ∼73 Å over time. Scale bar: x, y, 100 nm; z, 10 nm.

Figure 5

Determination of residues in close proximity to the membrane surface in both prepore and pores by collisional quenching. The extent by which the fluorescence from selected labeled residues is reduced in bilayers that contain lipids with a quenching moiety in the headgroup region (TEMPO-PC), compared with the fluorescence from similarly modified proteins in bilayers without the TEMPO-PC (POPC), is shown. The residues denoted on the X-axis were mutated to cysteine and labeled with the fluorescent probe in the disulfide-locked mutant, PFOS190C-G57C. The extent of quenching was determined in both prepore and pore complexes of each dye-labeled mutant. Fluorophores located near the surface of the membrane surface will be quenched by the TEMPO-PC present in the liposomes. Legend: POPC−DTT, PFOS190C-G57C incubated with POPC-cholesterol liposomes as a prepore complex (disulfide remains oxidized); POPC+DTT, PFOS190C-G57C incubated with POPC-cholesterol liposomes as a pore complex (disulfide is reduced to allow prepore-to-pore conversion); TEMPO−DTT and TEMPO+DTT (same as POPC−DTT and POPC+DTT, except that 10% of the total lipid is replaced with TEMPO-labeled lipid). F/F0, ratio of fluorescence of membrane-bound PFO (F) to that for the soluble monomer (F0).

Figure 6

Location of residues near the N-terminus of PFO of the prepore complex. The amino-terminal residues of PFO, as well as the amino-terminal polyhistidine tag, are predicted to be near the top of the oligomeric complex. The locations of the histidine tag and residue D30C were each identified in the oligomeric complex by specifically tagging each with a large protein and imaging with AFM. (A) The addition of antibodies that recognize the amino-terminal polyhistidine epitope near the N-terminus results in the appearance of a number of large globular particles on the surface of the oligomers. The particles in the middle region were scraped away by the application of a greater force prior to obtaining this image. The difference in height between the middle and the unperturbed region is ∼10 nm, which is consistent with the size of antibodies (Han et al, 1995). Scale bar: x, y, 100 nm; z, 25 nm. (B) Addition of a thiol-reactive biotin label to PFOY181A/D30C prepore complex (left panel) followed by the addition of streptavidin (right panel) results in the appearance of a number of ∼8 nm globular particles (right image) that are distributed in a pattern which resembles the surfaces the prepore complexes. Vertical scale: 20 nm. Scale bar: 100 nm.

Figure 7

Location of residues near the N-terminus of PFO in the pore complex. Addition of the same antibodies described for Figure 6A produces a change in the sample topography in the PFO pore complexes (left image) as found with the prepore complexes. Likewise, following the same biotinylation/streptavidin labeling procedure as in Figure 6B with the pore-forming mutant PFOD30C produced a similar change in topography in the pore complexes (right image) as observed with prepore complexes. Thus, residues near the N-terminus are located at the top of both prepore and pore complexes and are similarly accessible to these structural labels in both complexes. Scale bar: 100 nm.

Figure 8

Schematic structural model of the prepore-to-pore transition of PFO. The data presented here suggest that the conformation of PFO in the prepore complex is largely the same as that of the water-soluble monomer, attached to the membrane in a perpendicular orientation via the tip of its domain 4. At the prepore stage of formation, the TMHs are ∼40 Å from the membrane surface. Upon converting to the pore, there is a vertical collapse of the structure by 40 Å, which changes neither the outer diameter of the complex nor the structure of its topmost surface significantly. We propose that the vertical collapse is a consequence of a disruption of the extended domain 2 structure. As a result of this collapse, the TMHs are brought close enough to the membrane surface to be able to span the bilayer and line the pore.