Structural elements of the cholesterol-dependent cytolysins that are responsible for their cholesterol-sensitive membrane interactions

Abstract

The pore-forming mechanism of the cholesterol-dependent cytolysins (CDCs) exhibits an absolute requirement for membrane cholesterol. The structural elements of the CDCs that mediate this interaction are not well understood. Three short hydrophobic loops (L1-L3) and a highly conserved undecapeptide sequence at the tip of domain 4 of the CDC structure are known to anchor the CDC to the membrane. It has been thought that the undecapeptide directly mediates the interaction of the CDCs with a cholesterol-rich cell surface. Herein we show that the L1-L3 loops, not the undecapeptide, are responsible for mediating the specific interaction of the CDCs with cholesterol-rich membranes. The membrane insertion of the undecapeptide was uncoupled from membrane binding by the covalent modification of the undecapeptide cysteine thiol. Modification of the cysteine prevented prepore to pore conversion, but did not affect membrane binding, thus demonstrating that undecapeptide membrane insertion follows that of the L1-L3 loops. These studies provide an example of a structural motif that specifically mediates the interaction of a bacterial toxin with a cholesterol-rich membrane.

Fig. 1.

The crystal structure of ILY and the domain-4 crystal structures of ILY and PFO. (a) A ribbon representation of the crystal structure of ILY (34) denoting the positions of various structures and residues referred to in this work. (b) An overlay of a ribbon representation of the D4 structures of ILY (pink) and PFO (blue) based on the crystal structures of both proteins (34, 44). Shown are the locations of the undecapeptide and the L1–L3 loop residues of ILY and PFO (the PFO loop residues are in parentheses). The images were generated by using Visual Molecular Dynamics (45).

Fig. 2.

The ILY undecapeptide residue A486 inserts into cholesterol-depleted membranes. ILY residue Ala-486 was mutated to a cysteine (ILY^A486C) and derivatized with NBD. The fluorescence emission of the NBD was determined for ILY^A486C-NBD incubated alone (solid line), with hRBCs (dashed line), or with hRBCs depleted of cholesterol (dotted line).

Fig. 3.

L1, L2, and L3 of ILY do not insert into cholesterol-depleted membranes. Each D4 loop was substituted with a cysteine, modified with NBD, and the fluorescence emission was independently determined for each in the absence or presence of native or cholesterol-depleted hRBC ghost membranes. (a–c) ILY^A428C-NBD (a), ILY^A464C-NBD (b), or ILY^L518C-NBD (c) was incubated alone (solid line), with hRBCs (dashed line), or with hRBCs depleted of cholesterol (dotted line). (d–f) Membrane cholesterol was then restored, and insertion was determined for ILY^A428C-NBD (d), ILY^A464C-NBD (e), or ILY^L518C-NBD (f) alone (solid line) or after incubation with cholesterol-replete membranes (dotted line).

Fig. 4.

The L1–L3 loops mediate PFO binding to cholesterol-rich liposomes. Shown is SPR-binding analysis of aspartate- and glycine-substituted PFO loop mutants for residues Ala-401 (loop L2), Ala-437 (loop L3), and Leu-491 (loop L1). (a) SPR-detected binding of native PFO (solid line), PFO^A401D (dashed line), and PFO^A401G (dotted line). (b) SPR-detected binding of native PFO (solid line), PFO^A437D (dashed line), and PFO^A437G (dotted line). (c) SPR-detected binding of native PFO (solid line), PFO^L491D (dashed line), and PFO^L491G (dotted line). RU, resonance units.

Fig. 5.

Chemical modification of the undecapeptide cysteine of PFO does not prevent binding to cholesterol-rich liposomes. (a) FRET between PFO^C459-Alexa and unlabeled liposomes (solid line) or rhodamine-PE-labeled liposomes (dashed line). (b) The SPR-detected binding of native PFO (solid line) and native PFO modified at the native undecapeptide cysteine (Cys-459) with NEM (PFO^NEM) (dashed line). RU, resonance units.

Fig. 6.

Modification of the PFO undecapeptide cysteine thiol blocks membrane insertion of the undecapeptide tryptophans and conversion of the prepore to pore. (a) The intrinsic emission intensity of the tryptophans in native PFO increases as it moves from solution (solid line) to its membrane-bound state (dashed line). The emission of the tryptophans was quenched by the inclusion of the membrane-restricted collisional quencher 7-DOXYL (dotted line). (b) The experiments shown in a were repeated with native PFO that was modified at Cys-459 with NEM. (c) SDS/AGE analysis of oligomer formation by native PFO and NEM-modified PFO.

Fig. 7.

Schematic summary of the effects on pore formation of cholesterol and oxidation of the undecapeptide cysteine thiol. ILY and PFO D4 domains are depicted with membranes (gray). (Upper) After ILY binds to hCD59, the L1–L3 loops insert into the membrane in a cholesterol-dependent manner. This insertion is followed by the cholesterol-independent insertion of the undecapeptide residue Ala-486 of the undecapeptide with the subsequent formation of the pore. It is not known whether the tryptophan residues of the ILY undecapeptide insert into the membrane (they are not shown as inserted in the model). In the absence of cholesterol, Ala-486 of ILY inserts into the membrane after receptor binding, but loops L1–L3 do not, thus trapping ILY in the prepore complex. (Lower) Loops L1–L3, the undecapeptide tryptophan residues, and Cys-459 of PFO insert into the membrane in cholesterol-rich membranes. Preventing the insertion of any single L1–L3 loop prevents PFO binding to cholesterol-rich membranes, similar to the effect seen with membranes that lack cholesterol. Oxidation or modification of the Cys-459 thiol blocks membrane insertion of the undecapeptide tryptophan residues and traps PFO in the prepore complex.