DISULFIDE BRIDGES OF A CYSTEINE-RICH REPEAT OF THE LDL RECEPTOR LIGAND-BINDING DOMAIN

The low density lipoprotein (LDL) receptor is the prototype of a famil y of structurally related cell surface receptors that mediate the endo cytosis of multiple ligands in mammalian cells. Its ligand-binding dom ain consists of seven cysteine-rich ligand-binding repeats, each simil ar to 40 amino acid residues long. Ligand-binding repeats occur in oth er members of the LDL receptor (LDLR) gene family and in a number of f unctionally unrelated proteins. As a first step toward an understandin g of the structure and function of LB repeats, we have expressed the a mino-terminal ligand-binding repeat (LB1) of the human LDLR as a recom binant peptide (rLB1) and have determined its disulfide-pairing scheme . Oxidative folding of rLB1 yielded a single isomer which contained th ree disulfide bonds. This isomer reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDLR, suggestin g that rLB1 was correctly folded. rLB1 was resistant to digestion with trypsin, chymotrypsin, and V8 protease, consistent with a tightly fol ded structure. Disulfide bond connections were established using two s eparate approaches. Digestion with the nonspecific proteolytic enzyme proteinase K yielded an 8 amino acid peptide with a single disulfide b ond which connected Cys(IV) and Cys(VI). In the second approach, disul fide bonds were sequentially reduced with tris(2-carboxyethyl)phosphin e and the resulting cysteine residues alkylated with iodoacetamide. An analysis of peptides which contained two cysteinylacetamide residues, derived from a single reduced disulfide bond, showed that Cys(I) and Cys(III) were disulfide-bonded and confirmed the presence of a disulfi de bond between Cys(IV) and Cys(VI). We infer that the remaining disul fide bond bridges Cys(II) and Cys(V). These disulfide bonds result in a cluster of negatively-charged residues, including the conserved Ser- Asp-Glu sequence, in a single loop, in place for interactions with pos itively charged residues on apoB-100 and apoE. The disulfide bond conn ectivity of rLB1 1 serves as a paradigm for other members of the LB fa mily.