[Show abstract][Hide abstract] ABSTRACT: The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40-45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB(1-2)) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB(1-2) has now been solved using two-dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and H alpha chemical shifts and the low phi and psi angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB(1-2) structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.
[Show abstract][Hide abstract] ABSTRACT: The ligand-binding domain of the low-density lipoprotein (LDL) receptor is comprised of seven tandemly repeated ligand-binding modules, each being approximately 40 amino acids long and containing six conserved cysteine residues. We have expressed and characterized a concatemer of the first two modules (LB1 and LB2) of the human LDL receptor. Oxidative folding of the recombinant concatemer (rLB1-2), in the presence of calcium ions, gave a single dominant isomer with six disulfide bonds. Peptic cleavage of the short linker region that connects the last cysteine residue of LB1 and the first cysteine residue of LB2 yielded two discrete fragments, thus excluding the presence of intermodule disulfide bonds. The N-terminal module, LB1, reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDL receptor. From this, we concluded that the first module was correctly folded, with the same set of disulfide bonds as LB1 of the LDL receptor. The disulfide bond connections of LB2 were identified from mass spectral analysis of fragments formed by digestion of the C-terminal peptic fragment with elastase. These data showed that the disulfide bonds of LB2 connected Cys(I) and Cys(III), Cys(II) and Cys(V), and Cys(IV) and Cys(VI). This pattern is identical to that found for recombinant LB1 and LB2. The concatemer has two high-affinity calcium-binding sites, one per module. An analysis of the secondary chemical shifts of Calpha protons shows that the conformations of LB1 and LB2 in the concatemer are very similar to those of the individual modules, with no evidence for strong interactions between the two modules.
[Show abstract][Hide abstract] ABSTRACT: The ligand-binding domain of the low-density-lipoprotein (LDL) receptor comprises seven cysteine-rich repeats, each approximately 40 amino acids long. The deletion of two amino acids (Asp26 and Gly27) from the first of these repeats (LB1), leads to a defective LDL receptor, and the clinical syndrome of familial hypercholesterolemia [Leitersdorf, E., Hobbs, H. H., Fourie, A. M., Jacobs, M., van der Westhuyzen, D.R. & Coetzee, G.A. (1988) Proc. Natl Acad. Sci. USA 85, 7912-7916]. Receptors which reach the cell surface fail to bind IgG-C7, a conformation-specific monoclonal antibody directed to LB1. To determine the effects of the two-amino-acid deletion on the folding of the LB1 of the LDL receptor, we have expressed LB1 and the mutant repeat, des-Asp26, Gly27-LB1, as recombinant (rLB1 and des-Asp26, Gly27-rLB1) peptides, and have determined their ability to fold in vitro. Unlike rLB1, which folded into a single isomer that was recognized by IgG-C7 and had three disulfide bonds, des-Asp26, Gly27-rLB1 folded into an equilibrium mixture of four isomers. Each of these isomers contained three disulfide bonds, but none were recognized by IgG-C7. We suggest that LDL receptors in the endoplasmic reticulum (ER) of the cell also fold into an equilibrium mixture of distinct receptor molecules, each with an abnormally folded isomer of des-Asp26, Gly27-LB1, and that the retarded transport of receptors to the cell surface arises because only a subset of the isomers reaches the cell surface.
Full-text · Article · Aug 1996 · European Journal of Biochemistry
[Show abstract][Hide abstract] 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)
[Show abstract][Hide abstract] ABSTRACT: The human LDL receptor (LDLR) has a binding domain which consists of seven contiguous ligand-binding (LB) repeats, each approximately 40 amino acids long with three disulfide bonds. The second LB repeat, which is required for full binding of LDL, has been expressed, purified and folded to yield a single, fully oxidized isomer. By selective reduction and alkylation, we have shown that the cysteine residues have a I-III, II-V, IV-VI connectivity, matching that recently determined for the amino-terminal repeat. We suggest that the first two LB repeats of the LDLR, with their unique disulfide-bonding pattern, serve as a structural paradigm for other LB repeats.