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

摘要

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 cys-teine 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.