Structure of the LDL Receptor Extracellular Domain at Endosomal pH

Abstract

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 (1,10,11). Further, the LDL-R cysteine-rich repeats contain clusters of acidic residues implicated not only in ligand binding but also in calcium binding, as shown at pH ϭ 5.0 crystallographically by Fass et al. (12) and at pH ϭ 6.7 with the use of nuclear magnetic resonance (NMR) imaging (13). The calcium ions enable the repeats to bind to the ligand (14,15).Dissociation of ligands is crucial for receptor recycling and hence prop...

Apolipoprotein E isoform-specific binding to the low-density lipoprotein receptor

“…The ability to monitor receptor-ligand interactions in real time indicates this system is amenable to determination of association and dissociation kinetic parameters with the additional potential to investigate the effect of mutations in LDLR on ligand binding and/or release. Given the availability of an X-ray structure for sLDLR [22] and lipid-free apoE-NT, a detailed understanding of the molecular determinants of the binding interaction of this physiologically important ligand may be achieved in this system. Such studies should reveal the molecular properties responsible for the isoform-and lipid-dependent effects on apoE binding to LDLR family members.…”

Versatility in ligand recognition by LDL receptor family proteins: advances and frontiers

“…Other structures containing inter-and intramolecular binding interfaces between LA repeats and their partners also share the essential elements of the recognition mode seen in the LA3-4/RAP-D3 complex ( Figure 3). These complexes include the long-range intramolecular interface between LA repeats four and five and the YWTD beta-propeller domain [14], and a VLDLR fragment bound to human rhinovirus serotype 2 (HRV2), for which the LDLR and VLDLR serve as the primary receptor for cell entry [15]. In the structure of the giant erythrocruorin respiratory complex from the earthworm Lumbricus terrestris, interactions between the LA repeats of three linker chains with the hemoglobin b subunits of the complex also exhibit this canonical binding mode with a minor variation: the basic side chains projecting into the acidic pocket are from arginine residues, rather than from lysines [16].…”

“…This binding mode also appears to be tailored for a multivalent kind of recognition, because of the small area contributed by each LA repeat to the interface. Thus, two repeats are used in the LA3-4/RAP-D3 interface, multirepeat fragments exhibit much higher affinity for the HRV2 capsid than single repeats [15], and the LA4-5 pair of repeats is needed to establish the longrange interface with the propeller domain at endosomal pH [14].…”

“…The LDLR is in an open, binding-active conformation at neutral pH, but adopts a closed, binding inactive conformation at low pH with a long-range interface between the propeller domain and LA repeats four and five [14]. Biochemical studies implicate three histidine residues at the interface between the propeller domain and LA repeats four and five in reversibly modulating the conformation of the LDLR as a function of pH [9].…”

A functional transmembrane complex: The luteinizing hormone receptor with bound ligand and G protein

“…The relaxin-binding receptors exhibit one low-density lipoprotein (LDL) receptor "class A" sequence motif in their N-terminal domains that is absent in the other members of the GPHR cluster (Hsu et al, 2000), although a related snail (Lymnaea stagnalis) LRR-containing receptor has many LDL receptor "class A" motifs (Tensen et al, 1994). This sequence motif was modeled using an LDL receptor "class A" motif from the crystal structure of the human LDL receptor (Rudenko et al, 2002). Representative models, shown in Fig.…”