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Incomplete pneumolysin oligomers form membrane pores.

Sonnen AF, Plitzko JM, Gilbert RJ - Open Biol (2014)

Bottom Line: Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores.We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores.As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.

View Article: PubMed Central - PubMed

Affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.

ABSTRACT
Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.

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Implications for pore formation by CDCs. Schematic of a pore formed by a ring of pneumolysin subunits (top left), by an arc of subunits with a toroidal lipid edge (top right) and a close-up of the toroidal structure, as also found in pores formed by electroporation and by proteins such as Bax, equinatoxin II and colicin E1 [3,37–42] and truncated α-haemolysin [43].
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RSOB140044F5: Implications for pore formation by CDCs. Schematic of a pore formed by a ring of pneumolysin subunits (top left), by an arc of subunits with a toroidal lipid edge (top right) and a close-up of the toroidal structure, as also found in pores formed by electroporation and by proteins such as Bax, equinatoxin II and colicin E1 [3,37–42] and truncated α-haemolysin [43].

Mentions: Data on a variety of other pore structures indicate that proteolipid pores—consisting of matrices of protein and lipid—do exist and are characterized by a toroidal arrangement of the lipids themselves, as shown in figure 5. This solution to pore formation by CDCs was first proposed in 1985 by Bhakdi et al. [20] and supported by their later work which showed how truncated (arc) oligomers of streptolysin form functional pores of reduced size [19]. Experimental evidence that such a lipid arrangement is possible now comes from a variety of sources, including X-ray diffraction studies of the α5 helical peptide derived from pro-apoptotic Bax [37], viscoelastic studies of membranes with the bee-venom peptide melittin [44], transbilayer lipid dynamics in the presence of the Xenopus antimicrobial peptide magainin [45], NMR and FTIR studies of the sea anemone protein equinatoxin II [38] and the effect that lipids promoting toroidal lipid structures have on colicin E1 pore formation [39]. The toroidal form of lipid structure is also expected during electroporation [40] and to exist during membrane fusion [46], which suggests that it can persist for sufficient lengths of time to play a significant role in CDC activity. A recent single-particle reconstruction of the proteolipid pores formed by full-length Bax [41] and imaging analysis of Bax pores formed in giant unilamellar vesicles over periods of hours [42] further demonstrate that the lifetime of such structures is sufficient for a biologically relevant effect. Both α-helix-based (as in Bax) and β-sheet-based (as in CDCs) mechanisms of pore formation appear capable of proteolipid pore formation; a recent study showed this even for the canonical β-barrel pore-forming protein α-haemolysin [43] while another recent report described simulations using β-sheet arcs of protegrin which supported the formation of pores via a toroidal lipidic-structure-based mechanism [47].Figure 5.


Incomplete pneumolysin oligomers form membrane pores.

Sonnen AF, Plitzko JM, Gilbert RJ - Open Biol (2014)

Implications for pore formation by CDCs. Schematic of a pore formed by a ring of pneumolysin subunits (top left), by an arc of subunits with a toroidal lipid edge (top right) and a close-up of the toroidal structure, as also found in pores formed by electroporation and by proteins such as Bax, equinatoxin II and colicin E1 [3,37–42] and truncated α-haemolysin [43].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043118&req=5

RSOB140044F5: Implications for pore formation by CDCs. Schematic of a pore formed by a ring of pneumolysin subunits (top left), by an arc of subunits with a toroidal lipid edge (top right) and a close-up of the toroidal structure, as also found in pores formed by electroporation and by proteins such as Bax, equinatoxin II and colicin E1 [3,37–42] and truncated α-haemolysin [43].
Mentions: Data on a variety of other pore structures indicate that proteolipid pores—consisting of matrices of protein and lipid—do exist and are characterized by a toroidal arrangement of the lipids themselves, as shown in figure 5. This solution to pore formation by CDCs was first proposed in 1985 by Bhakdi et al. [20] and supported by their later work which showed how truncated (arc) oligomers of streptolysin form functional pores of reduced size [19]. Experimental evidence that such a lipid arrangement is possible now comes from a variety of sources, including X-ray diffraction studies of the α5 helical peptide derived from pro-apoptotic Bax [37], viscoelastic studies of membranes with the bee-venom peptide melittin [44], transbilayer lipid dynamics in the presence of the Xenopus antimicrobial peptide magainin [45], NMR and FTIR studies of the sea anemone protein equinatoxin II [38] and the effect that lipids promoting toroidal lipid structures have on colicin E1 pore formation [39]. The toroidal form of lipid structure is also expected during electroporation [40] and to exist during membrane fusion [46], which suggests that it can persist for sufficient lengths of time to play a significant role in CDC activity. A recent single-particle reconstruction of the proteolipid pores formed by full-length Bax [41] and imaging analysis of Bax pores formed in giant unilamellar vesicles over periods of hours [42] further demonstrate that the lifetime of such structures is sufficient for a biologically relevant effect. Both α-helix-based (as in Bax) and β-sheet-based (as in CDCs) mechanisms of pore formation appear capable of proteolipid pore formation; a recent study showed this even for the canonical β-barrel pore-forming protein α-haemolysin [43] while another recent report described simulations using β-sheet arcs of protegrin which supported the formation of pores via a toroidal lipidic-structure-based mechanism [47].Figure 5.

Bottom Line: Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores.We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores.As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.

View Article: PubMed Central - PubMed

Affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.

ABSTRACT
Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.

Show MeSH