Figure 5 : Lytic activity of ILY variants.

From: Disentangling the roles of cholesterol and CD59 in intermedilysin pore formation

Figure 5

(a) ILY crystal structure (PDB ID: 1S3R)9 with domains colored as in Fig. 2. Residues mutated in ILY variants shown as spheres. (b) ILY variants were incubated with calcein-containing liposomes decorated with cytoCD59, unless otherwise indicated by cytoCD59. All liposomes contain cholesterol unless otherwise indicated by –Cholesterol. Fluorescence measurements, expressed as a percent total lysis, were normalized against background and detergent burst vesicles. Error bars indicate standard deviations across three independent experiments. (c) Schematic illustrating the temporal roles of CD59 and cholesterol in ILY pore formation. ILY and CD59 are represented as space-filled models from the crystal structure of the complex (PDB ID: 4BIK). ILY D3 is in lilac; the remainder of the structure is in blue; CD59 is green. Soluble ILY is targeted to cholesterol-containing membranes (red sphere), whereby CD59 sets a specific geometry that defines the diameter of an oligomeric ILY prepore (blue 3D barrel). CD59 triggers the vertical collapse of this prepore, enabling the transmembrane segments of D3 (lilac) to approach the bilayer. CD59 is released from an SDS-resistant late prepore prior to membrane insertion, while cholesterol is required for the final membrane perforation step.