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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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Schematic of pore formation by CDCs (and MACPF proteins). In the left-hand column, we show the atomic structure of perfringolysin [23] (top) with the helices forming transmembrane hairpins on pore formation coloured red and the regions involved in membrane binding cyan and blue; and a model of the pre-pore (middle) and pore (bottom) with the transmembrane β-hairpins deployed. In the central column, we show schematic diagrams for monomers binding to the membrane (top), forming a pre-pore (middle) and a pore (bottom). To the right, we show in grey the subunit profiles of the pre-pore and pore states, which are distinctively different. The pre-pore has a comma-like profile very similar to the atomic model of perfringolysin, for example [23], while the pore has a more compact and thinner profile.
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RSOB140044F1: Schematic of pore formation by CDCs (and MACPF proteins). In the left-hand column, we show the atomic structure of perfringolysin [23] (top) with the helices forming transmembrane hairpins on pore formation coloured red and the regions involved in membrane binding cyan and blue; and a model of the pre-pore (middle) and pore (bottom) with the transmembrane β-hairpins deployed. In the central column, we show schematic diagrams for monomers binding to the membrane (top), forming a pre-pore (middle) and a pore (bottom). To the right, we show in grey the subunit profiles of the pre-pore and pore states, which are distinctively different. The pre-pore has a comma-like profile very similar to the atomic model of perfringolysin, for example [23], while the pore has a more compact and thinner profile.

Mentions: The mechanism of pore formation for CDCs has been defined to date with respect to the larger pores formed by full rings of oligomerized subunits (figure 1). Their conversion from a water-soluble to a membrane-inserted form requires the refolding of a set of α-helices into a pair of transmembrane β-hairpins (TMHs), creating a continuous β-sheeted wall to the pore [24,25]. This process occurs only once the formation of a pre-pore oligomeric ring is completed [26,27] and involves a doubling-over of the CDC subunit in order to bring the TMHs to an appropriate position to span the membrane bilayer [28,29]. Is this mechanism of pore formation able to accommodate a role for incomplete rings? As previously discussed [3,18,30], it can because all that need differ between a full-ring and an arc forming a pore through a membrane is the point at which membrane insertion occurs, and this is probably governed by a kinetic mechanism and relates to the CDC concentration and membrane fluidity.Figure 1.


Incomplete pneumolysin oligomers form membrane pores.

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

Schematic of pore formation by CDCs (and MACPF proteins). In the left-hand column, we show the atomic structure of perfringolysin [23] (top) with the helices forming transmembrane hairpins on pore formation coloured red and the regions involved in membrane binding cyan and blue; and a model of the pre-pore (middle) and pore (bottom) with the transmembrane β-hairpins deployed. In the central column, we show schematic diagrams for monomers binding to the membrane (top), forming a pre-pore (middle) and a pore (bottom). To the right, we show in grey the subunit profiles of the pre-pore and pore states, which are distinctively different. The pre-pore has a comma-like profile very similar to the atomic model of perfringolysin, for example [23], while the pore has a more compact and thinner profile.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140044F1: Schematic of pore formation by CDCs (and MACPF proteins). In the left-hand column, we show the atomic structure of perfringolysin [23] (top) with the helices forming transmembrane hairpins on pore formation coloured red and the regions involved in membrane binding cyan and blue; and a model of the pre-pore (middle) and pore (bottom) with the transmembrane β-hairpins deployed. In the central column, we show schematic diagrams for monomers binding to the membrane (top), forming a pre-pore (middle) and a pore (bottom). To the right, we show in grey the subunit profiles of the pre-pore and pore states, which are distinctively different. The pre-pore has a comma-like profile very similar to the atomic model of perfringolysin, for example [23], while the pore has a more compact and thinner profile.
Mentions: The mechanism of pore formation for CDCs has been defined to date with respect to the larger pores formed by full rings of oligomerized subunits (figure 1). Their conversion from a water-soluble to a membrane-inserted form requires the refolding of a set of α-helices into a pair of transmembrane β-hairpins (TMHs), creating a continuous β-sheeted wall to the pore [24,25]. This process occurs only once the formation of a pre-pore oligomeric ring is completed [26,27] and involves a doubling-over of the CDC subunit in order to bring the TMHs to an appropriate position to span the membrane bilayer [28,29]. Is this mechanism of pore formation able to accommodate a role for incomplete rings? As previously discussed [3,18,30], it can because all that need differ between a full-ring and an arc forming a pore through a membrane is the point at which membrane insertion occurs, and this is probably governed by a kinetic mechanism and relates to the CDC concentration and membrane fluidity.Figure 1.

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