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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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Classification of two-dimensional projections of sub-tomogram averages. (a) The main images show class averages of the projections and display complete rings as well as arcs (incomplete rings) of protein. The insets in each case are the corresponding statistical I-image, calculated using IMAGIC software [33] and as defined in the Material and methods (above), and the class variance (below). The number of projections found in each class is also given on each average and the bar in the first image indicates 250 Å. (b) The members of each of the classes shown in panel (a); the class average is shown again as the final image in each montage, and the bar in the first montage indicates 500 Å.
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RSOB140044F2: Classification of two-dimensional projections of sub-tomogram averages. (a) The main images show class averages of the projections and display complete rings as well as arcs (incomplete rings) of protein. The insets in each case are the corresponding statistical I-image, calculated using IMAGIC software [33] and as defined in the Material and methods (above), and the class variance (below). The number of projections found in each class is also given on each average and the bar in the first image indicates 250 Å. (b) The members of each of the classes shown in panel (a); the class average is shown again as the final image in each montage, and the bar in the first montage indicates 500 Å.

Mentions: Purified pneumolysin was added to cholesterol-containing liposomes and incubated at 37°C for 5 s or 1 min prior to the collection of cryo-electron tomograms (see Material and methods for a full description of data acquisition and analysis). Longer incubation times result in sample aggregation; this phenomenon has not yet been explained but has been observed for pneumolysin using both spectroscopic and imaging methods and relates to a tendency of targeted membranes to bleb [28,31,32]. Following reconstruction (see the electronic supplementary material, Movies S1 and S2 for examples), we interactively selected from 34 different tomograms 1953 sub-volumes containing oligomers, taking care to choose examples seen both in profile and with the oligomeric ring viewed from above in the frame of reference. Projection images of top-view sub-volumes, when classified using IMAGIC software (see Material and methods) [33], clearly demonstrated the presence of rings and arcs of protein density both in the class averages and the statistical I-images, while the variances of each image showed no significant signal (see Material and methods) (figure 2a). Furthermore, the individual images from each class match well with their corresponding class average (figure 2b). This exercise suggested that both complete and incomplete oligomers were present in the sample and could be resolved from one another, and we therefore decided to perform three-dimensional volume classification and averaging, making use of a recently developed maximum-likelihood methodology [34].Figure 2.


Incomplete pneumolysin oligomers form membrane pores.

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

Classification of two-dimensional projections of sub-tomogram averages. (a) The main images show class averages of the projections and display complete rings as well as arcs (incomplete rings) of protein. The insets in each case are the corresponding statistical I-image, calculated using IMAGIC software [33] and as defined in the Material and methods (above), and the class variance (below). The number of projections found in each class is also given on each average and the bar in the first image indicates 250 Å. (b) The members of each of the classes shown in panel (a); the class average is shown again as the final image in each montage, and the bar in the first montage indicates 500 Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140044F2: Classification of two-dimensional projections of sub-tomogram averages. (a) The main images show class averages of the projections and display complete rings as well as arcs (incomplete rings) of protein. The insets in each case are the corresponding statistical I-image, calculated using IMAGIC software [33] and as defined in the Material and methods (above), and the class variance (below). The number of projections found in each class is also given on each average and the bar in the first image indicates 250 Å. (b) The members of each of the classes shown in panel (a); the class average is shown again as the final image in each montage, and the bar in the first montage indicates 500 Å.
Mentions: Purified pneumolysin was added to cholesterol-containing liposomes and incubated at 37°C for 5 s or 1 min prior to the collection of cryo-electron tomograms (see Material and methods for a full description of data acquisition and analysis). Longer incubation times result in sample aggregation; this phenomenon has not yet been explained but has been observed for pneumolysin using both spectroscopic and imaging methods and relates to a tendency of targeted membranes to bleb [28,31,32]. Following reconstruction (see the electronic supplementary material, Movies S1 and S2 for examples), we interactively selected from 34 different tomograms 1953 sub-volumes containing oligomers, taking care to choose examples seen both in profile and with the oligomeric ring viewed from above in the frame of reference. Projection images of top-view sub-volumes, when classified using IMAGIC software (see Material and methods) [33], clearly demonstrated the presence of rings and arcs of protein density both in the class averages and the statistical I-images, while the variances of each image showed no significant signal (see Material and methods) (figure 2a). Furthermore, the individual images from each class match well with their corresponding class average (figure 2b). This exercise suggested that both complete and incomplete oligomers were present in the sample and could be resolved from one another, and we therefore decided to perform three-dimensional volume classification and averaging, making use of a recently developed maximum-likelihood methodology [34].Figure 2.

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