ABSTRACT
Oligomers of pneumolysin form transmembrane channels in cholesterol-containing lipid bilayers. The mechanism of pore formation involves a multistage process in which the protein, at first, assembles into a ring-shaped complex on the outer-bilayer leaflet. In a subsequent step, the complex inserts into the membrane. Contrary to most investigations of pore formation that have focussed on protein changes, we have deduced how the lipid-packing order is altered in different stages of the pore-forming mechanism. An optical tweezing apparatus was used, in combination with microfluidics, to isolate large-unilamellar vesicles and control exposure of the bilayer to pneumolysin. By monitoring Raman-scattered light from a single-trapped liposome, the effect of the protein on short-range order and rotational diffusion of lipids could be inferred from changes in the envelope of the C-H stretch. A significant change in the lipid-packing order takes place during assembly of pre-pore oligomers. We were not able to detect a change in the lipid-packing order during the initial stage of protein binding, or any further change during the insertion of oligomers. Pre-pore complexes induce a transformation in which a bilayer, resembling a liquid-ordered phase is changed into a bilayer resembling a fluid-liquid-disordered phase surrounding ordered microdomains enriched in cholesterol and protein complexes.
Subject(s)
Cholesterol/metabolism , Streptococcus pneumoniae/metabolism , Streptolysins/chemistry , Streptolysins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cholesterol/chemistry , Hemolysis , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Microfluidic Analytical Techniques , Models, Molecular , Mutation , Optical Tweezers , Protein Binding , Spectrum Analysis, Raman , Streptolysins/genetics , Unilamellar Liposomes/chemistry , Unilamellar Liposomes/metabolismABSTRACT
A spectroscopic technique is presented that is able to identify rapid changes in the bending modulus and fluidity of vesicle lipid bilayers on the micrometer scale, and distinguish between the presence and absence of heterogeneities in lipid-packing order. Individual unilamellar vesicles have been isolated using laser tweezers and, by measuring the intensity modulation of elastic back-scattered light, changes in the biophysical properties of lipid bilayers were revealed. Our approach offers unprecedented temporal resolution and, uniquely, physical transformations of lipid bilayers can be monitored on a length scale of micrometers. As an example, the deformation of a membrane bilayer following the gel-to-fluid phase transition in a pure phospholipid vesicle was observed to take place across an interval of 54 ± 5 ms corresponding to an estimated full-width of only ~1 m°C. Dynamic heterogeneities in packing order were detected in mixed-lipid bilayers. Using a ternary mixture of lipids, the modulated-intensity profile of elastic back-scattered light from an optically-trapped vesicle revealed an abrupt change in the bending modulus of the bilayer which could be associated with the dissolution of ordered microdomains (i.e., lipid rafts). This occurred across an interval of 30 ± 5 ms (equivalent to ~1 m°C).
ABSTRACT
Pneumolysin is a cholesterol-dependent cytolysin (CDC) and virulence factor of Streptococcus pneumoniae. It kills cells by forming pores assembled from oligomeric rings in cholesterol-containing membranes. Cryo-EM has revealed the structures of the membrane-surface bound pre-pore and inserted-pore oligomers, however the molecular contacts that mediate these oligomers are unknown because high-resolution information is not available. Here we have determined the crystal structure of full-length pneumolysin at 1.98 Å resolution. In the structure, crystal contacts demonstrate the likely interactions that enable polymerisation on the cell membrane and the molecular packing of the pre-pore complex. The hemolytic activity is abrogated in mutants that disrupt these intermolecular contacts, highlighting their importance during pore formation. An additional crystal structure of the membrane-binding domain alone suggests that changes in the conformation of a tryptophan rich-loop at the base of the toxin promote monomer-monomer interactions upon membrane binding by creating new contacts. Notably, residues at the interface are conserved in other members of the CDC family, suggesting a common mechanism for pore and pre-pore assembly.
