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.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Bacteriocins/chemistry , Cholesterol/chemistry , Cytotoxins/chemistry , Hemolysin Proteins/chemistry , Membranes, Artificial , Amino Acid Substitution , Aspartic Acid/chemistry , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Bacteriocins/genetics , Crystallography, X-Ray , Cysteine/chemistry , Cytotoxins/genetics , Glycine/chemistry , Hemolysin Proteins/genetics , Liposomes/chemistry , Porosity , Protein Conformation , Tryptophan/chemistryABSTRACT
Three short hydrophobic loops and a conserved undecapeptide at the tip of domain 4 (D4) of the cholesterol-dependent cytolysins (CDCs) mediate the binding of the CDC monomers to cholesterol-rich cell membranes. But intermedilysin (ILY), from Streptococcus intermedius, does not bind to cholesterol-rich membranes unless they contain the human protein CD59. This observation suggested that the D4 loops, which include loops L1-L3 and the undecapeptide, of ILY were no longer required for its cell binding. However, we show here that membrane insertion of the D4 loops is required for the cytolysis by ILY. Receptor binding triggers changes in the structure of ILY that are necessary for oligomerization, but membrane insertion of the D4 loops is critical for oligomer assembly and pore formation. Defects that prevent membrane insertion of the undecapeptide also block assembly of the prepore oligomer, while defects in the membrane insertion of the L1-L3 loops prevent the conversion of the prepore oligomer to the pore complex. These studies reveal that pore formation by ILY, and probably other CDCs, is affected by an intricate and coupled sequence of interactions between domain 4 and the membrane.
