Proteins of the low-density lipoprotein receptor (LDLR) family are remarkable in their ability to bind an extremely diverse range of protein and lipoprotein ligands, yet the basis for ligand recognition is poorly understood. Here, we report the 1.26 A X-ray structure of a complex between a two-module region of the ligand binding domain of the LDLR and the third domain of RAP, an escort protein for LDLR family members. The RAP domain forms a three-helix bundle with two docking sites, one for each LDLR module. The mode of recognition at each site is virtually identical: three conserved, calcium-coordinating acidic residues from each LDLR module encircle a lysine side chain protruding from the second helix of RAP. This metal-dependent mode of electrostatic recognition, together with avidity effects resulting from the use of multiple sites, represents a general binding strategy likely to apply in the binding of other basic ligands to LDLR family proteins.

The low-density lipoprotein (LDL) receptor transports two different classes of cholesterol-carrying lipoprotein particles into cells: LDL particles, which contain a single copy of apolipoprotein B-100 (apoB-100), and beta-migrating very low-density lipoprotein (beta-VLDL) particles, which contain multiple copies of apolipoprotein E (apoE). The ligand-binding domain of the receptor lies at its amino-terminal end within seven adjacent LDL-A repeats (LA1-LA7). Although prior work clearly establishes that LA5 is required for high-affinity binding of particles containing apolipoprotein E (apoE), the number of ligand-binding repeats sufficient to bind apoE ligands has not yet been determined. Similarly, uncertainty exists as to whether a single lipid-activated apoE receptor-binding site within a particle is capable of binding to the LDLR with high affinity or whether more than one is required. Here, we establish that the LA4-5 two-repeat pair is sufficient to bind apoE-containing ligands, on the basis of binding studies performed with a series of LDLR-derived "minireceptors" containing up to four repeats. Using single chain multimers of the apoE receptor-binding domain (N-apoE), we also show that more than one receptor-binding site in its lipid-activated conformation is required to bind to the LDLR with high affinity. Thus, in addition to inducing a conformational change in the structure of N-apoE, lipid association enhances the affinity of apoE for the LDLR in part by creating a multivalent ligand.

Low-density lipoprotein (LDL) receptors bind lipoprotein particles at the cell surface and release them in the low pH environment of the endosome. The published structure of the receptor determined at endosomal pH reveals an interdomain interface between its beta propeller and its fourth and fifth ligand binding (LA) repeats, suggesting that the receptor adopts a closed conformation at low pH to release LDL. Here, we combine lipoprotein binding and release assays with NMR spectroscopy to examine structural features of the receptor promoting release of LDL at low pH. These studies lead to a model in which the receptor uses a pH-invariant scaffold as an anchor to restrict conformational search space, combining it with flexible linkers between ligand binding repeats to interconvert between open and closed conformations. This finely tuned balance between interdomain rigidity and flexibility is likely to represent a shared structural feature in proteins of the LDL receptor family.