Membrane Interactions of Pneumolysin.

In the prepore conformation, PLY interacts with membranes via the undecapeptide and loop 1 (L1) of D4. To characterize the membrane interaction of PLY and account for its cholesterol dependence, we performed all-atom MD simulations of PLY monomers adhered via D4 to model bilayers consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with or without cholesterol. In a bilayer with 30% cholesterol, the PLY monomer remained bound to the upper leaflet for the entire duration of the simulation (8 μs; Fig. 2A and SI Appendix, Fig. S1). By contrast, in the absence of cholesterol, we observed unbinding and dissociation of PLY from the membrane surface (after 0.5 μs; Fig. 2B). At higher cholesterol levels, the Trp-rich undecapeptide (residues Trp433, Trp435, and Trp436) along with L1 residues (Thr459, Leu460, Tyr461) interacted with cholesterol molecules directly but transiently (Fig. 2C and SI Appendix, Fig. S2). Peripheral membrane binding often triggers conformational changes in proteins (28). In the case of PLY, the undecapeptide residues Trp435 and Trp436 started out detached but then rearranged to anchor the protein into the bilayer (SI Appendix, Fig. S2). Hinge bending around D2 dominates the large-scale dynamics of the membrane-docked monomer (SI Appendix, Fig. S3). This motion has previously been associated with the transition of PLY to the so-called late prepore state (15, 19).

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Fig. 2.

Membrane interaction of PLY protomers. (A and B) Snapshots from all-atom MD simulations of PLY protomers that were initiated from structures docked to bilayers with and without cholesterol. (C) Zoom-in on membrane interaction in A, showing the Trp-rich undecapeptide and loop 1 in contact with a cholesterol molecule (highlighted spheres).

Coarse-grained MD simulations using the MARTINI model confirmed the docking sites and cholesterol dependence observed in atomistic MD simulations. PLY monomers docked spontaneously to the upper leaflet, using the undecapeptide and L1 residues (SI Appendix, Fig. S4). In simulations with mutations in undecapeptide and L1 residues, we observed fewer docking events and longer waiting times (SI Appendix, Fig. S5A). By contrast, cholesterol aided the docking of PLY to DOPC bilayers (SI Appendix, Fig. S5B). Despite its severely restricted backbone dynamics, the coarse-grained model thus captures key membrane interactions identified in our all-atom MD simulations.

Wetting and Lipid Depletion Near Pneumolysin.

To test the impact of PLY on membrane structure, we performed all-atom MD simulations of PLY with membrane-inserted β-hairpins. In MD simulations of the monomer, we found that the transmembrane β-sheet formed by four antiparallel β-strands was strongly distorted, including the β-strands in D3. The high flexibility in D2 allowed D4 to tilt from an upright into a flatter position, loosening the contacts of L1 with the membrane. In one of two replicates, the β-sheet was completely pushed out of the membrane and PLY dislodged from the membrane (within 1 μs; SI Appendix, Fig. S6). In this process, first D1 and D3 were lifted. Once the tips of the β-hairpins lost contact with the polar head groups of the lipids, they quickly crossed the membrane. The tips of the bent β-sheet lay almost flat on the membrane. In oligomer simulations, we found that the outer β-strands of the transmembrane β-sheet of the PLY trimer (SI Appendix, Fig. S7) and of a PLY pentamer (Fig. 3) were also severely distorted into bent conformations. By contrast, the central β-strands of protomers in the pentamer were not distorted in a major way. Lateral cross-protomer H-bonds thus stabilize the β-sheet arcs upon insertion into the membrane (Fig. 3).

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Fig. 3.

Lipids recede from the hydrophilic inner surface of the transmembrane β-sheet formed by PLY oligomers in pore conformation. Shown are three snapshots from an all-atom MD simulation of an arc-shaped PLY pentamer embedded in a DOPC bilayer with 30% cholesterol, as seen from above (A–C) and along the transmembrane β-sheet (D–F). After water (red dots) is drawn to the inner face of the β-sheet (purple strands), the lipid molecules recede and form an open membrane edge that allows free exchange of lipids between the upper and lower leaflets (B and E). Water influx expands the gap into a membrane pore between the protein and the nascent bilayer edge. See SI Appendix, Figs. S6 and S7 for monomer and trimer simulations, respectively, and Movie S1.

The transmembrane β-sheet formed by PLY oligomers in pore conformation has an outer hydrophobic face and an inner hydrophilic face, one attracting and the other repelling lipid tails. Within a few nanoseconds in the atomistic pentamer MD simulation, the inner face became hydrated. Water influx triggered lipid reorientation (Movie S1). Within 10 ns, the lipids facing the hydrophilic side of the β-sheets formed an open membrane edge with loosely packed lipids (Fig. 3E). The region between the edge and the β-sheet was devoid of lipids and allowed free exchange of water and ions across the membrane. In PLY-trimer simulations (SI Appendix, Fig. S7), distorted transmembrane arcs produce a narrow water channel. The zone of lipid clearance was more pronounced in the pentamer simulation (Fig. 3). The β-sheets of the pentamer arranged into a compact half-moon shape that remained stable throughout the following 1-μs simulation time with only one of the outer protomers being distorted (SI Appendix, Fig. S8A). The corresponding lipid fingerprint (29) shows that cholesterol preferably interacted with D4, whereas the lipid-adhered region on the outside of the inserted β-sheets was almost devoid of cholesterol, as was the free membrane edge (SI Appendix, Fig. S8 B and C). The inner face of the β-sheets preferentially interacted with cations (here, sodium; SI Appendix, Fig. S9) via a membrane-spanning region of acidic residues. While the monomer facilitated membrane entry and passage of only a few sodium ions (SI Appendix, Fig. S9 A and B), the trimer formed a persistent pore of about 2 nm diameter (SI Appendix, Fig. S9 C and D). The pentamer pore with its free membrane edge was already too large to be cation selective (SI Appendix, Fig. S9 E and F). These simulations provide an atomistically detailed view of the changes in membrane structure induced by fully inserted β-sheets and thus into the behavior of lipids, water, and ions during pore formation.

Lipid Escape from PLY Rings.

To resolve the pore formation by complete PLY rings with 42 monomers, we performed MD simulations with the MARTINI coarse-grained model (30⇓–32). We modeled different β-sheet insertion states as putative intermediates along a sequential insertion pathway, from a ring entirely in prepore conformation (0/42) to a fully inserted ring (42/42; Fig. 4 A–F and Movies S2–S4). In the MD simulations, the lipid tails quickly receded from the inner face of inserted β-sheet segments while water entered, and a membrane edge formed (Fig. 4 A–F), consistent with the observations from our atomistic simulations for small oligomers.

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Fig. 4.

Pore formation by pneumolysin. (A–F) Top views of final frames of independent coarse-grained simulations of PLY rings with increasing fraction of protomers in pore conformation (orange to red) inserted in DOPC bilayers (gray) with cholesterol (yellow). (G–I) Snapshots (cross-section) from simulation showing the effect of partial insertion (50% insertion, as in C) in a setup allowing free lipid flow out of the PLY ring via the lower leaflet. In this setup, the presence of an equivalent pore on the other leaflet (SI Appendix, Fig. S13) largely eliminates back pressure against lipid efflux. (J–L) Snapshots (cross-section) from simulation initiated from completely inserted PLY ring (red) as in F. The enclosed bilayer patch, constrained by the ring, forms a closed vesicle. In a cell, we expect that this vesicle will be expelled by osmotic flow, leaving a large cytolytic pore.

The enclosed membrane patches deformed to minimize the length of the exposed free edge. For all rings with more than 25% of the PLY β-hairpins inserted, we observed measurable changes in curvature of the enclosed bilayer patch (SI Appendix, Fig. S10). A small kink below adhered prepore protomers left the bilayer intact and continuous with the outside membrane (Fig. 4G, right). With all 42 protomers in pore conformation, the transmembrane β-sheet formed a cylindrical wall (Fig. 4 F and J–L). Lacking a connection to the outside membrane, the enclosed membrane patch detached from the hydrophilic wall and vesiculated (Fig. 4F).

Already in prepore conformation, fully formed PLY rings restrict the flow of lipids in and out of the ring. By tracing the motion of enclosed lipids, we found that the PLY undecapeptide and L1 in prepore conformation act as barriers for lipid diffusion in the upper leaflet. By contrast, lipids in the lower leaflet moved freely and mixed with the bulk (SI Appendix, Fig. S11). However, even though the lipids from the upper leaflet of the enclosed membrane patch could slide across the open edge and escape via the lower leaflet, this route is effectively blocked in our simulation setup. The reason is that such lipid efflux would build up pressure in the lower leaflet as a result of growing leaflet asymmetry. In effect, our setup traps the lipid plug (Fig. 4 G–I), allowing us to probe pore opening when β-sheet insertion is completed rapidly or when lipid efflux is hindered.

To overcome the restriction of lipid flow under periodic boundary conditions, we doubled the system size, simulating two equivalent rings adhered on opposite sides of the bilayer. The leaflet asymmetries built up by lipid efflux from the two rings exactly compensate, thus allowing the enclosed lipids to escape via the open edges (Fig. 4 G–I and SI Appendix, Figs. S11 and S13). The initially curved membrane plugs relaxed into a flatter configuration (SI Appendix, Fig. S10) as excess lipids drained out of the rings. In simulations of PLY rings with a greater fraction of protomers in pore conformation, the transmembrane β-hairpins restricted lateral lipid diffusion out of the ring and increased the relaxation times required for leaflet equilibration, leading to a slower decay of curvature of the enclosed bilayer patches (SI Appendix, Fig. S10).

Validation by Experiment.

The simulations make several concrete and testable predictions: Pore formation does not proceed to completion if not all protomers have inserted their helix bundles as β-hairpins, lipids attach to the prepore PLY segments in the ring, and the membrane buckles within pores that restrict lipid escape. In a survey of images from earlier AFM studies (15), we found several structures that closely resemble key intermediate configurations observed in our simulations (SI Appendix, Figs. S14–S16). In particular, we identified examples (SI Appendix, Fig. S14) for (i) complete prepores and (ii) complete pores. We also found (iii) a partially inserted ring with an arc of prepore conformations and an arc of pore conformations and (iv) a pore ring containing what appears to be a trapped lipid blob. The structures iii and iv are consistent with the simulation structures in Fig. 4 H, I, K, and L, respectively, as intermediate configurations with fractional insertions and a completely inserted ring with vesiculating lipids. The membrane deformations induced by the PLY ring are similar to those seen in recent cryo-EM experiments, both outside and inside the ring (10, 18, 19) (SI Appendix, Fig. S17).

Pore Opening by Vesiculation.

Elastic theory (33) predicts that for small PLY ring radii, a fully detached membrane patch trapped inside a ring maintains an open-disk shape, whereas for large radii the line tension on the detached rim induces a spontaneous vesiculation of the patch. Using elastic theory (SI Appendix), we computed the energy for membrane shapes ranging from flat bilayer disks to spherical vesicles for a given PLY inner-ring radius R0 (SI Appendix, Fig. S18). A similar transition occurs also for partially inserted pores (SI Appendix, Fig. S19). With values for the bending rigidity and line tension typical of plasma membranes, the transition between the two regimes occurs at R0≈10 nm, i.e., the typical size of PLY rings (10). Consistent with this estimate, prepore rings tend to have radii below R0, and pore-conformation rings with open pores have larger radii, as seen in AFM and EM experiments (SI Appendix, Fig. S18B, Bottom).