title: Disulfide bridges of a cysteine-rich repeat of the LDL receptor ligand-binding domain.
abstract: The low density lipoprotein (LDL) receptor is the prototype of a family of structurally related cell surface receptors that mediate the endocytosis of multiple ligands in mammalian cells. Its ligand-binding domain consists of seven cysteine-rich ligand-binding repeats, each approximately 40 amino acid residues long. Ligand-binding repeats occur in other members of the LDL receptor (LDLR) gene family and in a number of functionally unrelated proteins. As a first step toward an understanding of the structure and function of LB repeats, we have expressed the amino-terminal ligand-binding repeat (LB1) of the human LDLR as a recombinant peptide (rLB1) and have determined its disulfide-pairing scheme. Oxidative folding of rLB1 yielded a single isomer which contained three disulfide bonds. This isomer reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDLR, suggesting that rLB1 was correctly folded. rLB1 was resistant to digestion with trypsin, chymotrypsin, and V8 protease, consistent with a tightly folded structure. Disulfide bond connections were established using two separate approaches. Digestion with the nonspecific proteolytic enzyme proteinase K yielded an 8 amino acid peptide with a single disulfide bond which connected Cys(IV) and Cys(VI). In the second approach, disulfide bonds were sequentially reduced with tris(2-carboxyethyl)phosphine 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 disulfide bond between Cys(IV) and Cys(VI). We infer that the remaining disulfide bond bridges Cys(II) and Cys(V).(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Queensland, Brisbane, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Bieri
authors.forename: J T
authors.initials: JT
authors.lastname: Djordjevic
authors.forename: N L
authors.initials: NL
authors.lastname: Daly
authors.forename: R
authors.initials: R
authors.lastname: Smith
authors.forename: P A
authors.initials: PA
authors.lastname: Kroon
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D011973:Receptors, LDL / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D004220:Disulfides; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011973:Receptors, LDL; D011994:Recombinant Proteins; D003545:Cysteine