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

Abstract

Perfringolysin O (PFO), a bacterial cholesterol-dependent cytolysin, binds 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 three short α-helices that unfold and ultimately refold into two transmembrane β-hairpin (TMH) components of the membrane-embedded β-barrel. Interstrand disulfide-bond scanning revealed that β-strands in a fully assembled PFO β-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 TMHs that are hydrogen bonded to those of adjacent subunits in the bilayer-embedded β-barrel.

Figure 1. PFO structure and structural alterations

(a) Individual domains of the elongated PFO monomer bound to a membrane surface (pale blue) are depicted in different colors (left), while the ribbon representation (right) shows the locations of TMH1 (magenta) and TMH2 (green) when folded into α-helices within domain 3 in monomeric PFO. The core β-sheet (gray) and four individual residues are also shown. The image was generated using Chimera (http://www.cgl.ucsf.edu/chimera). (b) The transition from helical TMHs in the PFO monomer to β-hairpins in the β-barrel pore is depicted. Four antiparallel β-strands of the D3 core that ultimately extend into the membrane (box) are identified as β1-β4 as shown. (c) When monomeric PFO binds to a membrane, β5 first rotates away from β4, and this then allowsβ4 to form hydrogen bonds with β1 from another PFO. β4 and β1 alignment relative to each other involves the π-stacking of an aromatic residue found in each strand (open rectangles). (d) The arrangement of atoms in anti-parallel β-strands is fixed by inter-strand hydrogen bonding. A C_α atom on one side of the β-sheet is separated by 5.5 Å from the juxtaposed C_α to which it is hydrogen bonded and by 7–9 Å from the next-nearest C_α atom on the adjacent β-strand. Since the two carbon atoms in a disulfide bond are separated by less than 4.5 Å, a disulfide bond can only be formed between the C_β atoms directly opposite each other in adjacent anti- parallel β-strands.

Figure 2. Detection of disulfide-bonded PFO dimers in PPC and pore complexes

(a) An equimolar mixture of S193C and Q308C PFO derivatives in pore complexes (incubated 40 min, 37°C) or PPCs (2 h, 4°C) were prepared in the presence of 100 μM Na-tetrathionate, extracted in some cases with methanol/chloroform, and analyzed by SDS-AGE with or without boiling prior to electrophoresis. The yield of disulfide-linked dimers is shown below the lane. (b) Dimer (193–308) formation in pore complexes was stimulated by 100 μM Na-tetrathionate or blocked by either 5 mM DTT or pre-treatment with 20 mM NEM. (c) Dimers were detected in pore complexes with other pairs of mono-cysteine PFO derivatives with or without tetrathionate. (d) No homodimers were observed in pore complexes formed with only a single mono-cysteine derivative.

Figure 3

β4-β1 cross-linking and β-strand alignment in pore complex. (a) Eachβ1 mutant was mixed with an equimolar amount of each β4 mutant in the presence of tetrathionate, and then incubated (40 min, 37°C) with cholesterol-rich liposomes to form pore complexes. Dimer formation was detected by SDS-AGE. (b) The % yield of disulfide-linked dimers was calculated from the intensities of the bands shown in (a): 100 × dimer/(dimer + monomer). The average yields (± S.D.) are shown for three or more independent experiments with each pair of β1 and β4 mutants. (c) Seven specific β1-β4 pairs formed a disulfide bond in the β-barrel pores with high efficiency. Aligning β1 and β4 of adjacent monomers based on these crosslinks reveals a two residue-offset in the tips of the two β-strands. (d) An untilted (α = 0°) parallel alignment of strands in a β-barrel is created when adjacent β-strands and TMHs are not offset. (e) When TMHs are offset by 2 residues as in panel c, the closed ends of the TMHs must tilt to form a circular β-barrel in the plane of the membrane.

Figure 4. Dimer formation in PPCs

(a) An equimolar mixture of S193C and Q308C rPFO mutants were incubated for 2 h at 4°C to form PPCs, and the effects of 100 μM sodium tetrathionate, 5 mM DTT, and pre-treatment with 20 mM NEM on disulfide crosslinking of PFO monomers are shown. (b) The tetrathionate dependence of disulfide crosslinking is shown for two other pairs of β4 and β1 mutants in PPCs. (c) In PPCs, the Q308C rPFO mutant forms homodimers in the presence of tetrathionate, while the S193C mutant does not. (d) Samples were prepared as in Figure 3a in the presence of tetrathionate, and then incubated (2 h, 4°C) to form PPCs. Dimer formation was detected by SDS-AGE. (e) The % yield of disulfide-linked dimers for each pair of β1 and β4 mutants was determined as in Figure 3b. Mean values (± S.D.) are shown for three or more independent experiments with each pair of β1 and β4 mutants.

Figure 5. Fluorescence-detected changes in TMH environment reveal stages in TMH unfolding and alignment during pore formation

(a) Fluorescence emission spectra are shown for NBDs positioned at each of four different sites in D3 (see Fig. 1a). Each sample was sequentially transitioned through three states: soluble monomer; membrane-bound PPC; and membrane-embedded pore complex. The spectra are normalized to accurately reflect the relative emission intensities, with one arbitrary unit (a.u.) defined as the peak intensity of the S193C monomer. (b) TMH1 (magenta) and TMH2 (green) helices folded around the core β-sheet (gray) in PFO monomers are converted to membrane-inserted hairpins in the pore complex β-barrel through any of several possible PPC structures. Since PPC TMHs are partially unfolded and not aligned, intermediate states with the TMHs folded into helices (top) or aligned in a β-sheet (bottom) do not occur. A few of the β4-β1 disulfide crosslinking sites observed in the presence of sodium tetrathionate are indicated by the dots in the TMHs shown in the center panel.