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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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Related in: MedlinePlus

Sub-tomogram class averages of pre-pore and partial pore structures observed in situ on liposomal membranes. Five different sub-classes representing pre-pores or partial pores, comprising in total 1065 sub-tomogram volumes, were identified in the whole dataset of 1953. They are shown viewed from above alone (first image from left) and with the pre-pore single-particle reconstruction [28] superimposed (second image), and from the side both as a whole and sectioned through the middle (upper and lower views in the third panel of each row). Protein is coloured red and membrane yellow. Maps varied in the completeness of the pre-pore oligomer (iv), the presence of a second membrane bilayer as in multilamellar liposomes ((ii) and (iii)) and the extent to which the membrane remained intact ((v) appears to be a transitioning partial pore, and (iv) may be also). The numbers of sub-volumes in each class map and their resolution together with the Fourier shell correlation (FSC) for each case are shown on the right-hand side.
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RSOB140044F3: Sub-tomogram class averages of pre-pore and partial pore structures observed in situ on liposomal membranes. Five different sub-classes representing pre-pores or partial pores, comprising in total 1065 sub-tomogram volumes, were identified in the whole dataset of 1953. They are shown viewed from above alone (first image from left) and with the pre-pore single-particle reconstruction [28] superimposed (second image), and from the side both as a whole and sectioned through the middle (upper and lower views in the third panel of each row). Protein is coloured red and membrane yellow. Maps varied in the completeness of the pre-pore oligomer (iv), the presence of a second membrane bilayer as in multilamellar liposomes ((ii) and (iii)) and the extent to which the membrane remained intact ((v) appears to be a transitioning partial pore, and (iv) may be also). The numbers of sub-volumes in each class map and their resolution together with the Fourier shell correlation (FSC) for each case are shown on the right-hand side.

Mentions: We subjected the whole dataset to iterative three-dimensional classification and alignment with the software XMipp (see Material and methods) [34]. We started with alignment to an average of all the sub-volumes and, taking care to keep the oligomers centred, ultimately identified 12 distinct class averages; in the paper, we show nine class maps from 1841 sub-volumes or 94% of the data (figures 3 and 4). The sub-tomographic class average maps were characterized by a planar structure from which ring- or arc-like density projected; the former was obviously identifiable as the bilayer surface, the latter as the pneumolysin oligomer. Some of the maps (figure 3, volumes (ii) and (iii) and figure 4, volumes (ii) and (iii)) had two lipid surfaces because they were associated with double-membraned vesicles (see the electronic supplementary material, Movies S1 and S2 also). Furthermore, while some of the pneumolysin oligomers were in a pre-pore state, others were in a pore-forming state, as indicated by the presence or absence of a membrane plugging the centre of the oligomeric ring [28]. However, as in the two-dimensional analysis (figure 2), the morphology of the pneumolysin oligomer varied between a partial ring (an arc) of protein and a full ring. We assessed the resolution of each sub-tomographic volume by Fourier shell correlation (figures 3 and 4) and they varied between 25 and 38 Å.Figure 3.


Incomplete pneumolysin oligomers form membrane pores.

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

Sub-tomogram class averages of pre-pore and partial pore structures observed in situ on liposomal membranes. Five different sub-classes representing pre-pores or partial pores, comprising in total 1065 sub-tomogram volumes, were identified in the whole dataset of 1953. They are shown viewed from above alone (first image from left) and with the pre-pore single-particle reconstruction [28] superimposed (second image), and from the side both as a whole and sectioned through the middle (upper and lower views in the third panel of each row). Protein is coloured red and membrane yellow. Maps varied in the completeness of the pre-pore oligomer (iv), the presence of a second membrane bilayer as in multilamellar liposomes ((ii) and (iii)) and the extent to which the membrane remained intact ((v) appears to be a transitioning partial pore, and (iv) may be also). The numbers of sub-volumes in each class map and their resolution together with the Fourier shell correlation (FSC) for each case are shown on the right-hand side.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140044F3: Sub-tomogram class averages of pre-pore and partial pore structures observed in situ on liposomal membranes. Five different sub-classes representing pre-pores or partial pores, comprising in total 1065 sub-tomogram volumes, were identified in the whole dataset of 1953. They are shown viewed from above alone (first image from left) and with the pre-pore single-particle reconstruction [28] superimposed (second image), and from the side both as a whole and sectioned through the middle (upper and lower views in the third panel of each row). Protein is coloured red and membrane yellow. Maps varied in the completeness of the pre-pore oligomer (iv), the presence of a second membrane bilayer as in multilamellar liposomes ((ii) and (iii)) and the extent to which the membrane remained intact ((v) appears to be a transitioning partial pore, and (iv) may be also). The numbers of sub-volumes in each class map and their resolution together with the Fourier shell correlation (FSC) for each case are shown on the right-hand side.
Mentions: We subjected the whole dataset to iterative three-dimensional classification and alignment with the software XMipp (see Material and methods) [34]. We started with alignment to an average of all the sub-volumes and, taking care to keep the oligomers centred, ultimately identified 12 distinct class averages; in the paper, we show nine class maps from 1841 sub-volumes or 94% of the data (figures 3 and 4). The sub-tomographic class average maps were characterized by a planar structure from which ring- or arc-like density projected; the former was obviously identifiable as the bilayer surface, the latter as the pneumolysin oligomer. Some of the maps (figure 3, volumes (ii) and (iii) and figure 4, volumes (ii) and (iii)) had two lipid surfaces because they were associated with double-membraned vesicles (see the electronic supplementary material, Movies S1 and S2 also). Furthermore, while some of the pneumolysin oligomers were in a pre-pore state, others were in a pore-forming state, as indicated by the presence or absence of a membrane plugging the centre of the oligomeric ring [28]. However, as in the two-dimensional analysis (figure 2), the morphology of the pneumolysin oligomer varied between a partial ring (an arc) of protein and a full ring. We assessed the resolution of each sub-tomographic volume by Fourier shell correlation (figures 3 and 4) and they varied between 25 and 38 Å.Figure 3.

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
Related in: MedlinePlus