Thirty years ago, Brown and Goldstein identified the low-density lipoprotein receptor (LDLR) in their search for the origin of the genetic disease familial hypercholesterolemia (FH) (Brown and Goldstein, 1986). FH-causing mutations of the gene encoding the LDLR disable receptor function in any of several different ways, each of which results clinically in elevated concentrations of plasma LDL and cholesterol. Investigation of the LDLR as a model system, facilitated in part by the use of FH-derived mutations, has repeatedly uncovered general insights about basic biological principles, including mechanisms for metabolic regulation, for receptor-mediated endocytosis, and for control of gene expression (Brown and Goldstein, 1986; Brown and Goldstein, 1997).

The physiologic role of the LDLR is to transport cholesterol-carrying lipoprotein particles into cells. The primary ligand for the receptor is low-density lipoprotein, or LDL, which contains a single copy of apolipoprotein B-100 (apoB); approximately 65-70% of plasma cholesterol in humans circulates in the form of LDL. The LDLR also binds tightly to beta-migrating forms of very low-density lipoprotein (b-VLDL), which contains multiple copies of apolipoprotein E (apoE). Receptor-ligand complexes enter the cell by endocytosis at clathrin-coated pits, where receptor molecules cluster on the cell surface. Bound lipoprotein particles are subsequently released in the low-pH milieu of the endosome, and the receptors then return to the cell surface in a process called receptor recycling.

The mature receptor is a modular type-1 transmembrane protein of 839 amino acids, and is representative of an entire class of receptors that contain LDL receptor type-A (LA), epidermal growth factor-like (EGF-like), and YWTD modules arranged in a similar pattern. The ectodomain of the receptor is functionally divided into two regions, a ligand-binding portion consisting of seven contiguous LDL-A modules (LA1-LA7) at the amino-terminal end, and a subsequent 400-residue region homologous to the EGF Precursor (EGFP). LA3 through LA7 are essential for binding of LDL; LA5 is also crucial for high-affinity binding of b-VLDL (Russell et al., 1989). The EGFP region, which encompasses two EGF-like modules, followed by a series of six YWTD repeats and a third EGF-like module, controls the related processes of lipoprotein release at low pH and recycling of the receptor to the cell surface. When this 400-amino acid piece of the receptor is deleted, apoE-containing ligands like b-VLDL still bind, but do not dissociate from the LDLR at endosomal pH, resulting in a receptor-recycling defect (Davis et al., 1987). More than half (54 %) of human FH point mutations lie in the EGFP domain, further highlighting the importance of the EGFP region in contributing to proper receptor function.

Initial structural studies of the LDLR focused on the ligand-binding modules, exploiting the modular organization of the receptor by using a protein dissection approach, in which individual domains and domain pairs were studied as isolated units. The NMR solution structure of LA1, a representative type A module, exhibited a novel fold with three disulfide bonds and little secondary structure. The high-resolution crystal structure of LA5, which we determined in collaboration with Debbie Fass and Jim Berger (Fass et al., 1997), revealed that four of the highly conserved acidic residues at the C-terminal end of the module stabilize the LDL-A fold by participating in the coordination of a calcium ion in an octahedral "cage" (Figure 1). The ion binding site explains the observed calcium requirement for proper folding and disulfide bond formation in LA modules, and provides a rationale for the known calcium requirement in lipoprotein binding. Many of the FH mutations within LA5 and other ligand-binding modules disrupt folding by directly altering calcium coordinating or key scaffolding residues that stabilize module structure.

Our structural studies of the EGFP region were facilitated by Tim Springer's prediction that the YWTD repeats would combine to fold into a single six-bladed b-propeller domain (Springer, 1998). In the X-ray crystal structure of a receptor fragment encompassing the YWTD domain and its two flanking EGF modules (Figure 2) we defined the structure of this propeller domain at high resolution, identified the mode of interaction of the C-terminal EGF-like module with the propeller, and enabled rationalization of the effects of many of the FH mutations located in the YWTD-EGF domain pair (Jeon et al., 2001). However, a model for how the propeller and its adjacent EGF-like modules might trigger release of ligands at endosomal pH was not readily apparent from the structure.

Low pH-induced ligand release and molecular logic of the LDLR.

The 3.7 Å resolution X-ray structure of the entire LDLR ectodomain at endosomal pH, determined recently by Deisenhofer's group in collaboration with Brown and Goldstein, now suggests a model for how the low pH environment of the endosome triggers release of lipoprotein ligands (Rudenko et al., 2002). The structure shows that ligand-binding modules 4 and 5, key contributors to LDL binding, are engaged in intramolecular contacts with the YWTD beta-propeller domain. This finding suggests a model in which the propeller domain of the receptor becomes an alternate ligand for the central lipoprotein-binding repeats in response to the drop in pH (Figure 3, animation).

One major unresolved mechanistic question is: does the propeller domain accelerate the rate of dissociation of bound LDL by actively displacing the ligand, or does the formation of the closed, low pH structure require prior dissociation of bound ligand? To distinguish between these two mechanistic possibilities, we plan to measure the rate of dissociation of bound LDL from the native receptor and compare it with the rate of dissociation of bound LDL from receptors lacking the beta-propeller domain (which we now know are functionally deficient in pH-mediated release of LDL). If the propeller domain actively displaces the ligand, then the complexes of native receptors with LDL should have a faster dissociation rate than the complexes with mutant receptors that lack the propeller domain.

Bibliography

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Brown, M. S., and Goldstein, J. L. (1997). The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89, 331-340.

Davis, C. G., Goldstein, J. L., Sudhof, T. C., Anderson, R. G., Russell, D. W., and Brown, M. S. (1987). Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature 326, 760-765.

Fass, D., Blacklow, S., Kim, P. S., and Berger, J. M. (1997). Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module. Nature 388, 691-693.

Jeon, H., Meng, W., Takagi, J., Eck, M. J., Springer, T. A., and Blacklow, S. C. (2001). Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair. Nat Struct Biol 8, 499-504.

Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989). Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins. J Biol Chem 264, 21682-21688.

Springer, T. A. (1998). An extracellular beta-propeller module predicted in lipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor precursor, and extracellular matrix components. J Mol Biol 283, 837-862.