Direct three-dimensional visualization of membrane disruption by amyloid fibrils.

Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer's, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from ß(2)-microglobulin (ß(2)m). Using cryoelectron tomography, we demonstrate that fragmented ß(2)m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15-25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid-protein interactions in membrane remodelling.

Structure of the poly-C9 component of the complement membrane attack complex.

The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming ß-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.

Structural basis of pore formation by the bacterial toxin pneumolysin.

The bacterial toxin pneumolysin is released as a soluble monomer that kills target cells by assembling into large oligomeric rings and forming pores in cholesterol-containing membranes. Using cryo-EM and image processing, we have determined the structures of membrane-surface bound (prepore) and inserted-pore oligomer forms, providing a direct observation of the conformational transition into the pore form of a cholesterol-dependent cytolysin. In the pore structure, the domains of the monomer separate and double over into an arch, forming a wall sealing the bilayer around the pore. This transformation is accomplished by substantial refolding of two of the four protein domains along with deformation of the membrane. Extension of protein density into the bilayer supports earlier predictions that the protein inserts beta hairpins into the membrane. With an oligomer size of up to 44 subunits in the pore, this assembly creates a transmembrane channel 260 A in diameter lined by 176 beta strands.