The low-density lipoprotein receptor mediates cholesterol homeostasis through endocytosis of lipoproteins. It discharges its ligand in the endosome at pH Ͻ 6. In the crystal structure at pH ϭ 5.3, the ligand-binding domain (modules R2 to R7) folds back as an arc over the epidermal growth factor precursor homology domain (the modules A, B,  propeller, and C). The modules R4 and R5, which are critical for lipoprotein binding, associate with the  propeller via their calcium-binding loop. We propose a mechanism for lipoprotein release in the endosome whereby the  propeller functions as an alternate substrate for the ligand-binding domain, binding in a calcium-dependent way and promoting lipoprotein release. The low-density lipoprotein receptor (LDL-R) regulates cholesterol homeostasis in mammalian cells. LDL-R removes cholesterolcarrying lipoproteins from plasma circulation in a process known as receptor-mediated endocytosis (1). Ligands bound extracellularly by LDL-R at neutral pH are internalized and then released in the endosomes ( pH Ͻ 6), leading to their subsequent lysosomal degradation. The receptor then recycles to the cell surface. Mutations in the LDL-R gene cause familial hypercholesterolemia (FH), one of the most common simply inherited genetic diseases (2). FH heterozygotes exhibit a reduced rate of receptor-mediated removal of plasma LDL by the liver, ultimately leading to early onset coronary heart disease and atherosclerosis. More than 920 mutations in LDL-R are known, some of which have been functionally characterized (2, 3). The extracellular domain of LDL-R is composed of a "ligand-binding domain" (with cysteine-rich repeats R1 to R7) and an "epidermal growth factor (EGF) precursor homology domain" (with the EGF-like repeats A, B, and C, as well as a  propeller between B and C) (4, 5). LDL-R binds LDL via the single protein in LDL, the 550-kD apolipoprotein B (apoB) (6); deleting R3, R4, R5, R6, or R7 reduces LDL binding to Ͻ20% of that of the wild-type LDL-R (7). LDL-R also binds to very low density lipoprotein (VLDL), -VLDL, intermediate density lipoprotein (IDL), and chylomicron remnants via the 33-kD apolipoprotein E (apoE) (8, 9); disrupting R5 decreases -VLDL binding to 30 to 50% of that of the wild-type receptor, whereas disrupting R4 or R6 reduces binding only slightly (7). At neutral pH, negative charges on repeats R1 to R7 are thought to interact with positive charges on apoB and apoE. Indeed, LDL binding to LDL-R can be disrupted competitively with polycations or permanently by selective chemical modification of positively charged residues on apoE or apoB Dissociation of ligands is crucial for receptor recycling and hence proper receptor function; mutations in LDL-R that impair ligand release produce FH (2). Deletion mutagenesis studies in LDL-R and the related VLDL-R have indicated that, although the ligand-binding domain is sufficient for binding lipoprotein particles, the receptor requires the EGF precursor homology domain for ligand release (16-18). The structural basis for LDL-R's ability to recognize a diverse group of lipoprotein particles, all varying in size, and release them at acidic pH is unknown. High-resolution crystal structures of modules R5 (12) and  propeller-C (5) are known, and solution NMR structures are known for single and tandem repeats, including R1, R2, R5, R6, A, and B Structure determination. The extracellular domain of human LDL-R (residues 1 to 699) was crystallized at pH ϭ 5.3, with the symmetry of space group P3 1 21 (28). Soaking crystals in sodium 12-tungstophosphate (Na 3 PW 12 O 40 ) improved their diffractive quality and incorporated large anomalous scatterers. The asymmetric unit contains a single protein molecule and two tungsten clusters as well as half of a tungsten cluster on a crystallographic twofold axis. Data collection, structure determination, and model statistics are given in Monomer description. There is clear electron density in the crystal structure for the modules R2, R3, R4, R5, R6, R7, A, B,  propeller, and C In the crystal, each monomer forms major contacts with five neighboring symmetry-related molecules. Although the relative orien
