Prepore to pore transition of a cholesterol-dependent cytolysin visualized by electron microscopy
Introduction
The cholesterol-dependent cytolysins (CDCs) constitute a large family of oligomerizing, pore-forming toxins from a variety of Gram-positive bacterial pathogens (reviewed in Rossjohn et al., 1999, Tweten et al., 2001). The CDCs belong to the superfamily of β-pore-forming toxins (β-PFTs) that includes such toxins as Staphylococcus aureus α-hemolysin and Bacillus anthracis anthrax toxin (Lacy and Stevens, 1998). The toxins produced by these bacteria are soluble monomers that bind to cell membranes via their respective receptors and oligomerize into pore-forming complexes. The anthrax toxin (Lacy et al., 2004, Petosa et al., 1997, Santelli et al., 2004) and α-hemolysin (Song et al., 1996) pore-forming complexes are each composed of seven monomers that form a pore of 1–3 nm. In contrast, CDC oligomers are comprised of up to 50 monomers and form an unusually large pore (25–30 nm in diameter) (Bhakdi et al., 1996, Czajkowsky et al., 2004, Gilbert et al., 1998, Morgan et al., 1995, Olofsson et al., 1993). The mechanisms by which water-soluble toxin complexes bind to the membrane and form transmembrane spanning pores have become the focus of immense interest in the last decade (Heuck et al., 2001, Zakharov and Crammer, 2002). To date the only toxin for which there exists an X-ray crystallographic structure resembling the oligomeric pore state is α-hemolysin (Song et al., 1996). To span the membrane this heptameric complex uses two β strands from each protomer to form a 14-stranded β-barrel. Similarly, a model based on the X-ray crystal structure of the water-soluble heptameric prepore of anthrax toxin suggests that a loop from each of its seven monomers rearranges to form the 14-stranded β-barrel that spans the membrane (Lacy et al., 2004, Petosa et al., 1997, Santelli et al., 2004).
The most studied CDCs are PFO, streptolysin O (SLO), and pneumolysin (PLY). These toxins exhibit significant similarity in their primary structures. Presently, PFO is the only member of the CDC family for which a crystal structure of the soluble monomer has been determined (Rossjohn et al., 1997). Similar to the other β-PFTs, the structure of the PFO monomer is rich in β-sheet and can be divided into four distinct contiguous domains (Fig. 1). To date, there are no X-ray crystal structures of the large oligomeric prepore or pore complexes from the CDC family. Oligomeric PLY complexes bound to cholesterol-containing liposomes have been studied by cryo-electron microscopy and image analysis (Gilbert et al., 1999, Gilbert et al., 2000). In these studies, the PLY protein monomers did not appear to fully penetrate the lipid bilayer. In contrast, extensive biochemical and biophysical studies indicate that the PFO protein monomers do span the membrane in the oligomeric pore complexes. The proposed sequence of events that gives rise to PFO pore formation is as follows. First, PFO monomers are anchored to the membrane by hydrophobic loops at the tip of domain 4 (Nakamura et al., 1998, Ramachandran et al., 2002). Next, monomers assemble into a membrane-bound prepore oligomeric ring (Hotze et al., 2001, Hotze et al., 2002). Finally, in a cooperative and rapid manner, the monomers undergo conformational changes that result in membrane insertion and pore formation. Spectroscopic fluorimetry studies of pore-forming PFO oligomers indicate that six short α-helices in domain 3 of each PFO monomer convert to form two transmembrane β-hairpins (designated TMH1 and TMH2 in Fig. 1) that give rise to an amphipathic β-barrel that spans the bilayer of the membrane (Hotze et al., 2001, Shatursky et al., 1999, Shepard et al., 1998). Interactions between domains 3 and 4 and the membrane appear to be coupled since mutations in the domain 3 hairpins that affect the rate of insertion of the β-barrel also affect interaction of domain 4 with the membrane (Heuck et al., 2000). It remains to be seen if other members of the CDCs follow a similar mechanistic pathway. In contrast to the models of the β-barrel pores of α-hemolysin and anthrax toxin which consist of 14 amphipathic β-strands, the β-barrel pore of PFO would be comprised of up to 100 β-strands.
The oligomeric rings of PFO are characterized by an outer and an inner ring of protein density. By comparing 2D projection maps from electron microscopy (EM) images of prepore PFO complexes and pore-forming PFO complexes we have shown that the intense and well-defined inner ring of density previously observed in EM images of CDCs on lipid membranes (Bhakdi et al., 1996, Gilbert et al., 1998, Morgan et al., 1995, Olofsson et al., 1993) is only associated with pore-forming complexes. In addition to the apparent conformational change from the prepore to the pore, we have identified specific domains of the toxin molecule within the image and have thus determined the orientation of PFO monomers within the oligomeric complex. These results are consistent with the current model for PFO membrane insertion and depict details of the molecular structure of a cholesterol-dependent cytolysin in its prepore and pore-forming complexes.
Section snippets
Preparation of C-terminally His-tagged PFO mutants
All mutants of PFO used herein were generated from the cysteine-less derivative of PFO, in which residue cysteine 459 was replaced by alanine (PFOC459A) as previously described (Czajkowsky et al., 2004, Hotze et al., 2002, Shepard et al., 1998). Recombinant PFO and its derivatives were expressed in E. coli and purified as described (Hotze et al., 2002).
Labeling of PFO with iodoacetyl-biotin or iodoacetamide fluorescein
The cysteine-substituted PFO mutants PFOS40C, dsPFOS171C and PFOK343C were generated from the disulfide-trapped derivative of PFOC459A in which
Formation of PFO oligomeric rings
The cysteine-free mutant PFOC459A used as a “wild-type” PFO control is capable of forming pores with wild-type levels of hemolytic activity (Shepard et al., 1998) and can form oligomeric rings on cholesterol-rich lipid layers similar to wild-type PFO (Olofsson et al., 1993) and to wild-type pore-forming pneumolysin (Gilbert et al., 1998, Morgan et al., 1995). The disulfide-trapped mutant PFOS190C-G57C (dsPFO) (Hotze et al., 2001) and the PFOY181A mutant (Hotze et al., 2002 JBC) which are devoid
Discussion
We have begun to characterize the conformational states of oligomeric rings of the cytolysin PFO on cholesterol-rich lipid layers by EM. Results from other biophysical studies of PFO show that, sometime between oligomerization and pore formation, six α-helices of domain 3 within each monomer restructure to form two transmembrane β-hairpins. This structural rearrangement is thought to involve a disordered intermediate state for domain 3. Presumably, in concert with this transition, the
Acknowledgments
We thank Dr. José Maria Carazo for the image processing package, XMIPP. We thank Amy Marpoe for technical assistance. Financial support for this research was provided by NIH 61938-01 (to E.M.W.K.) and NIH A1037657 (to R.K.T.).
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2019, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :For this reason, PFO is commonly considered a model CDC in studies that investigate the CDC pore-forming mechanism. The overall mechanism involved in the pore formation of several CDCs is relatively well characterized and requires several steps: recognition and binding to cholesterol; oligomerization of the soluble monomers on the membrane surface (prepore creation); and insertion of defined PFO regions into the lipid bilayer to form a large amphipathic transmembrane β-barrel pore with a diameter of 250–300 Å [12–15]. PFO is secreted by bacteria as a single, monomeric molecule that initially binds to cholesterol-containing membrane via a C-terminal lipid binding motif located in the D4 domain [16–19].
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2016, Current Opinion in Structural Biology
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Present address: Department of Botany and Microbiology, Oklahoma University, OK 73019, USA.
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Present address: The Burnham Institute, 10901 N. Torrey Pines Rd, La Jolla, CA 92037, USA.