Folding, Calcium Binding, and Structural Characterization of a Concatemer of the First and Second Ligand-Binding Modules of the Low-Density Lipoprotein Receptor

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 (rLB_1-2), 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 Cα 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.