Abstract
The plasma membrane provides an essential barrier, shielding a cell from the pressures of its external environment. Pore-forming proteins, deployed by both hosts and pathogens alike, breach this barrier to lyse target cells. Intermedilysin is a cholesterol-dependent cytolysin that requires the human immune receptor CD59, in addition to cholesterol, to form giant β-barrel pores in host membranes. Here we integrate biochemical assays with electron microscopy and atomic force microscopy to distinguish the roles of these two receptors in mediating structural transitions of pore formation. CD59 is required for the specific coordination of intermedilysin (ILY) monomers and for triggering collapse of an oligomeric prepore. Movement of Domain 2 with respect to Domain 3 of ILY is essential for forming a late prepore intermediate that releases CD59, while the role of cholesterol may be limited to insertion of the transmembrane segments. Together these data define a structural timeline for ILY pore formation and suggest a mechanism that is relevant to understanding other pore-forming toxins that also require CD59.
Highlights
That make up the interface are different from those that interact with cholesterol in the lipid bilayer[18,19]
CD59 is known to induce the formation of sodium dodecyl sulphate (SDS)-resistant ILY oligomeric pores on the surface of human cells[21]
For cholesterol-dependent cytolysins (CDCs) such as ILY, that require human CD59 in addition to cholesterol to form pores, structural transitions triggered by each component remain less clear
Summary
That make up the interface are different from those that interact with cholesterol in the lipid bilayer[18,19]. While it is known that both cholesterol and CD59 must be present for ILY to permeate the target cell, the precise role of each receptor remains unclear. We use model membrane systems decorated with CD59 and conformationally-locked ILY variants to disentangle structural transitions triggered by cholesterol and human CD59. We investigate the roles of each receptor in ILY oligomerization, vertical collapse of the prepore complex, and membrane lysis. Adopting a dual biochemical and biophysical approach, we find that CD59 is required for coordinating ILY monomers into an oligomeric prepore that can collapse towards the membrane. Formation of an SDS-resistant late prepore depends on structural transitions enabled by a movement between D2 and D3. Our data suggest that CD59 is released from the late-prepore, and that cholesterol triggers the final stages of membrane insertion
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