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The LDL receptor: how acid pulls the trigger
Review
TRENDS in Biochemical Sciences
Vol.30 No.6 June 2005
The LDL receptor: how acid pulls the trigger Natalia Beglova and Stephen C. Blacklow Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
The low-density lipoprotein receptor normally carries lipoprotein particles into cells, and releases them upon delivery to the low pH milieu of the endosome. Recent structural and functional studies of the receptor, combined with the plethora of prior knowledge about normal receptor function and the effects of diseaseassociated mutations that cause familial hypercholesterolemia, reveal a detailed molecular model for how the acidic environment of the endosome triggers the release of bound lipoprotein particles. Remarkably, the receptor dynamically interconverts between open (ligand-active) and closed (ligand-inactive) conformations in response to pH, relying on a specific arrangement of fixed and flexible interdomain connections to facilitate efficient binding and release of its lipoprotein ligands. Introduction The low-density lipoprotein receptor (LDLR) is responsible for transporting cholesterol-containing lipoprotein particles from the circulation into cells. Patients with lossof-function mutations in the LDLR gene have familial hypercholesterolemia (FH), an autosomal dominant genetic disorder affecting w1 in 500 individuals worldwide. In heterozygotes, FH is characterized clinically by elevated concentrations of plasma low-density lipoprotein (LDL) and cholesterol, and an increased risk of atherosclerosis and coronary heart disease. Untreated patients with homozygous FH have dramatically elevated levels of
plasma LDL and cholesterol, and typically die from heart disease at an early age [1]. The LDLR was first discovered by Brown and Goldstein in their search for the molecular basis underlying FH [2,3]. They found that fibroblasts from normal individuals bind LDL with high affinity and specificity, and suppress endogenous cholesterol synthesis when cultured in the presence of LDL. By contrast, cultured fibroblasts from homozygous FH patients did not suppress endogenous cholesterol synthesis and failed to take up LDL-cholesterol from the culture media. The gene encoding the LDLR was cloned and sequenced about ten years later [4,5] (GenBank accession number AH002776). Domain organization of the LDLR The mature LDLR is a modular, single-pass transmembrane glycoprotein of 839 amino acids (Figure 1). The seven adjacent LDL receptor type-A (LA) modules at the N-terminal end of the receptor are responsible for binding to lipoproteins [6]. Immediately C-terminal to these ligand-binding repeats is a region with homology to the epidermal growth factor precursor (EGFP), which consists of two epidermal growth factor-like (EGF) modules, followed by a YWTD domain and a third EGF module. This part of the LDLR is implicated in the release of bound lipoproteins at low pH [7]. Between the EGFP and the membrane is a region rich in serine and threonine residues that undergoes O-linked glycosylation. The
Key: LA repeat EGF-like module β-propeller domain O-linked sugar region NPxY endocytosis motif YxxL endocytosis motif
LDLR VLDLR ApoER2
LRP1 Megalin LRP5/6 Ti BS
Figure 1. Proteins of the LDLR family. The domain organization of the core family members is illustrated schematically. Corresponding author: Blacklow, S.C. ([email protected]). Available online 11 April 2005 www.sciencedirect.com 0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.03.007
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glycosylation region is believed to be a spacer separating the LDLR functional domains from the cell surface because a mutated LDLR lacking this spacer sequence is indistinguishable from the native receptor with respect to ligand binding, internalization and receptor recycling [8]. The O-linked sugar domain is then followed by a transmembrane segment and a 50-residue cytoplasmic tail, which is required for receptor localization in clathrincoated pits and for receptor endocytosis [9,10].
Cellular itinerary of the LDLR The pathway traversed by individual LDLR molecules in the cell is schematically illustrated in Figure 2. Immediately after synthesis, the precursor of the mature receptor migrates with an apparent molecular weight of 120 kDa on SDS–PAGE. Upon transport to the Golgi, the LDLR undergoes extensive O-linked glycosylation into the mature 160 kDa form found at the cell surface. Folding of the LDLR occurs in the ER in a non-vectorial manner [15,16]. Early in the folding process, the N-terminal cysteine-rich repeats form non-native disulfide bonds that reshuffle into native disulfide pairs, with the first LA repeat (LA1) acquiring its native fold late in the folding process [16]. The receptor associated protein (RAP), a common chaperone of LDLR family proteins, binds to the N-terminal LA repeats, facilitating their proper folding, www.sciencedirect.com
(3)
*
*
* *
*
*
Clathrincoated pit
(4)
*
*
Coated vesticles
*
*
Golgi *
LDLR family It is now evident that the LDLR constitutes the founding member of an entire class of transmembrane receptors (Figure 1). Members of the LDLR family conduct a wide range of physiologic functions, both as transporters of lipoprotein particles and other structurally unrelated ligands, and also as receptors or co-receptors in signal transduction cascades (see [11–14] for recent reviews). Although many proteins with LA, EGF or YWTD extracellular modules exist in the protein sequence database, each core member of the LDLR contains all three signature structural units of the LDLR. The most closely related family members are the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), which share w50% homology to the LDLR and to each other, and have an almost identical domain organization. These receptors, which are expressed in the brain, serve as receptors for a large secreted signaling protein called Reelin and have a crucial role in the migration of cortical neurons during development. Two giants of the family, LRP and megalin, have myriad and complex roles as both scavenger transporters and signaling molecules. Together, they constitute a second subgroup, in which the LA, EGF and YWTD modules are still arranged in a similar pattern. LRP-5 and LRP-6 still contain all three types of modules; however, the organization is different, with the YWTD domains preceding a series of three LA repeats adjacent to the membrane spanning domain. LRP-5 and LRP-6, and their homologues in frogs and flies, are co-receptors for Wnt signaling during development. Other distantly related receptors, with functions that are less welldefined, include LRP3, LRP9 and LRP12, which have LA and CUB domains in their extracellular portion, and SorLA-1 (LRP11).
LDL
LDLR
Recycling vesicle
(5)
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Endosome H+
*
(2)
* Lysosome Amino acids
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Cholesterol
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Ti BS
Figure 2. Cellular itinerary of the LDLR. (1) Receptors are first synthesized by ribosomes and folded in the endoplasmic reticulum. (2) Next, the receptors are glycosylated in the Golgi and transported to the cell surface. (3) At the plasma membrane, receptors bind lipoprotein ligands. (4) Internalization occurs via clathrin-coated pits, which ultimately deliver receptor–ligand complexes to endosomes. (5) After the bound lipoproteins are released, the receptors recycle back to the cell surface. Each of these 5 steps is also associated with a corresponding class of LDLR mutations found in FH. Initially adapted from Ref. [25], and modified, with permission, from Ref. [66] (www.els.net).
and preventing the premature binding of co-expressed ligands [17,18]. A second kind of chaperone protein, called Boca in Drosophila and Mesd in the mouse [19,20], is believed to assist folding of the YWTD b-propeller domains of LDLR family proteins [21]. Binding of circulating lipoproteins to the LDLR occurs at the cell surface. The most important physiological ligand for the LDLR is LDL, which contains a single copy of the apolipoprotein B-100 (apoB-100) as its primary protein component; 65–70% of plasma cholesterol in humans circulates in the form of LDL. In addition, the receptor also exhibits high-affinity binding of lipoproteins that contain multiple copies of apolipoprotein E (apoE), like b-migrating forms of very lowdensity lipoprotein (b-VLDL) and certain intermediate density lipoproteins [2,22]. Receptor-ligand complexes enter cells by endocytosis via clathrin-coated pits. Endocytosis of the LDLR requires the presence of an NPXY sequence in the cytoplasmic tail. Hobbs, Cohen and co-workers recently identified a gene that was mutated in an autosomal
Review
TRENDS in Biochemical Sciences
recessive form of FH [23]. The gene, which they called ARH-1, encodes a protein with a PTB domain capable of binding the NPXY sequence of the LDLR, a canonical clathrin-binding sequence LLDLE, and a sequence recognized by the b2-adaptin subunit of AP-2, the other major structural component of clathrin-coated pits [24]. These findings strongly suggest that ARH-1 serves as an adaptor protein that links the LDLR to the endocytic machinery by simultaneously binding to the NPXY sequence of the receptor cytoplasmic tail, clathrin and the AP-2 adaptor. After receptor–ligand complexes are taken up into clathrin-coated pits, they are then delivered to endosomes, where the low pH environment triggers release of the bound lipoprotein particles. Subsequently, the receptors return to the cell surface in a process called receptor recycling. By contrast, the released lipoprotein particles proceed to lysosomes, where the cholesterol esters are hydrolyzed to free cholesterol [25]. Remarkably, each LDLR cycles through its itinerary continuously, regardless of whether or not it is carrying bound cargo, completing each passage in 10–30 min. With a half-life of 16–24 h, a typical LDLR molecule recycles through 100 ‘acid baths’ or more before being degraded [26]. In patients with FH, more than 1000 mutations in the LDLR have been identified that interfere with one or more of these central events required for normal receptor function [27,28]. The mutations have been grouped into five classes, depending on what kind of receptor defect is observed (Figure 2). Thus, class 1 mutants fail to produce detectable amounts of protein; class 2 mutants have a partial or complete transport defect; class 3 mutants are impaired in ligand binding; class 4 mutants fail to localize in clathrincoated pits and are internalization-defective; and class 5 mutants exhibit a ligand release and recycling defect [1]. Mellman’s group recently showed that the G823D FH mutation, which lies toward the C-terminal end of the cytoplasmic tail beyond the NPXY sequence, interferes with proper sorting of the LDLR to the basolateral membrane of polarized epithelial cells, identifying yet another, sixth class of mutation implicated in FH [29].
Structure–function correlates in the LDLR ligand-binding domain Early mutational studies probing structure–function relationships in the LDLR showed that the LA repeats constitute the ligand-binding domain of the receptor [6]. Deletion of any repeat from LA3 to LA7, in addition to deletion of the first EGF repeat, interferes with the cell-surface binding of LDL. However, a recombinant form of the ligand-binding domain, including only the seven ligand-binding repeats, refolded after expression in bacteria, retains the ability to bind LDL, showing that the rest of the protein is not necessary for LDL binding once the protein is no longer on the cell surface [30]. Constraints on b-VLDL binding are less stringent because only deletion of LA5 effectively abrogates b-VLDL binding. Indeed, recent in vitro binding assays have identified the LA4-LA5 fragment of the LDLR as sufficient for binding apoE–DMPC complexes, which www.sciencedirect.com
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are believed to mimic b-VLDL with regard to receptor binding [31]. A picture of how the receptor recognizes lipoprotein ligands first began to emerge from structural studies of individual ligand-binding modules and module pairs, in combination with structural and biochemical studies of the receptor-binding domain of apoE. The NMR solution structure of the first ligand-binding repeat of the LDLR revealed a novel fold with three disulfide bonds and little secondary structure [32]. Studies investigating the requirements for formation of the native disulfide bonds of LA5 showed that calcium ions were required for proper folding, and that the bound calcium ion was required to maintain the structural integrity of the module [33]. Subsequently, the X-ray structure of LA5 identified four highly conserved acidic residues near the C-terminal end of the module, which stabilize the structure by participating in the coordination of a single bound calcium ion in an octahedral ‘cage’ [34]. The FH mutations in LA5 disrupt module folding by altering either the calcium coordinating residues or other key scaffolding residues in the module. In the absence of calcium, the structural integrity of the LA modules is lost and, as a result, the receptor fails to bind lipoprotein particles. NMR and crystallographic studies of other LA repeats, either individually (LA1, LA2, LA6, CR3, CR7, CR8 and TVA; reviewed in [32]; [35–41]), or in the context of module pairs, LA1–2, LA5–6 or LA7-EGF-A [42–44], also confirm the structural importance of calcium in specifying the LA module fold and are fully consistent with this interpretation of the role of calcium in LDL and b-VLDL binding. Another notable consequence of calcium coordination by the LA modules is that the conserved C-terminal acidic residues become clustered on one face of the module, creating a discrete patch of electronegative surface potential on each LA module. Both apoB and apoE, the protein moieties of LDL and b-VLDL, respectively, have a series of conserved basic residues that are required for binding to the LDLR, leading to the proposal that ligand recognition by the LDLR relies on electrostatic complementarity between receptor (acidic) and ligand (basic) [45,46]. One reason that it has been difficult to address this proposal at atomic resolution is that the receptor-binding domain of apoE requires association with lipid to become a high-affinity ligand for the LDLR [47]. Although the details of lipid activation are incompletely understood, the receptor-binding domain of apoE clearly changes its conformation upon lipid association. Before lipid activation, the receptor binding domain of apoE adopts an elongated four-helix bundle structure [48], whereas association with lipid causes the bundle to open up so that the hydrophobic interiors of the helices pack against the aliphatic tails of the lipids [49–52]. In the lipid-free form of apoE, the basic region required for binding to the LDLR lies on the solvent-exposed face of the fourth helix of the bundle, potentially in position to form contacts with the calcium-coordinating acidic clusters of the LA repeats. Why lipid activation is needed to enhance affinity, therefore, remains a puzzle. Perhaps, lipid association enhances the affinity for the receptor by inducing a specific curvature of the helices responsible for receptor
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recognition, by creating a multivalent ligand, by creating a composite protein-lipid surface required for recognition, or by some combination of these mechanisms. Role of flexible linkers connecting the LA repeats in facilitating ligand binding An unusual feature of the natural ligands for the LDLR is that they are highly heterogeneous in size and shape, posing a distinct challenge for recognition by a single receptor. Although LDL particles typically contain only a single copy ˚ in of apoB-100, they range in size from w180–270 A diameter [53–55]. b-VLDL particles have multiple copies of apoE per particle and are even larger, up to ˚ in diameter. 400 A How does the LDLR adjust its shape to bind different lipoprotein particles of varying size and composition? One important feature contributing to the versatility of the receptor in binding different ligands is that the linkers connecting adjacent ligand-binding modules seem to be flexible, even though all intermodule linkers, with the exception of the 12-residue linker connecting LA4 to LA5, are only 4–5 residues long. Studies of the LA1–LA2 and LA5–LA6 module pairs by solution NMR show that the linkers connecting the modules enable essentially unrestricted movement of one module with respect to the next in each case [42,56]. More generally, the absence of contacts between adjacent ligand-binding modules in the X-ray structure of the LDLR ectodomain (see section entitled Central role of the b-propeller in the intramolecular interactions at low pH) suggests that the rest of the ligand-binding modules are also unconstrained with respect to one another [57]. This intrinsic flexibility of the linkers connecting the ligand-binding modules, enabling relative freedom of motion for adjacent LA modules, permits the LDLR to accommodate its shape to bind a variety of heterogeneous lipoprotein particles. Structure and function of the EGFP domain The region of the LDLR that is implicated in release of bound lipoproteins at low pH is the epidermal growth factor precursor homology domain (EGFP). This part of the receptor lies immediately C-terminal to the ligandbinding domain and consists of two EGF-like modules (EGF-A and EGF-B), followed by a YWTD b-propeller domain and a third EGF-like module (EGF-C). When the entire EGFP region is deleted, the receptor is capable of binding b-VLDL and, to a lesser extent, LDL, but fails to release it at low pH [7,44,58]. The mutant receptor also fails to recycle efficiently and is degraded more rapidly than the native receptor after ligand binding [7]. The frequent occurrence of FH mutations in the EGFP region of the receptor also attests to its functional importance. Structural studies show that, in contrast to the LA modules of the ligand-binding domain, which are connected to one another by flexible linkers, some of the interdomain orientations of adjacent modules in the EGFP are fixed. The EGF-AB domain pair, solved independently by two different groups by solution NMR, adopts a rigid elongated conformation at neutral pH, with the interdomain orientation defined by calcium coordination at a site between the domains and by hydrophobic interdomain www.sciencedirect.com
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packing interactions [59,60]. The crystal structure of a receptor fragment containing the YWTD b-propeller (the fold of which was first predicted by Springer [61]) and its two flanking EGF modules, solved at neutral pH, revealed that the C-terminal EGF module (EGF-C) packs tightly against the b-propeller, whereas the EGF module preceding the propeller domain (EGF-B) is disordered in the crystal [62]. Elucidation of the structure enabled rationalization of the effects of many of the FH mutations located in the YWTD-EGF-C domain pair: one class of mutations alters side chains that form conserved packing and hydrogen-bonding interactions in the interior and between propeller blades, whereas a second subset of FH mutations are located at the interface between the propeller and EGF-C module, suggesting a structural requirement for maintaining the integrity of the interdomain interface. Although the X-ray structure provided important atomic details about interdomain relationships among modules within the EGFP region, and also about FH mutations, it did not immediately explain why the EGFP is required to promote the release of bound lipoproteins upon exposure to acidic pH.
Central role of the b-propeller in intramolecular interactions at low pH A key breakthrough that provided a structural rationale for the role of the EGFP domain in low-pH induced release of bound lipoproteins came when Deisenhofer’s group, in collaboration with Brown and Goldstein, determined the ˚ resolution crystal structure of the LDLR ectodomain to 3.7 A at endosomal pH [57]. In the low-pH structure, the receptor adopts a closed conformation, with a long-range intramolecular interface between the top face of the YWTD b-propeller and two of the central ligand-binding repeats, LA4 and LA5 (Figure 3). The closed structure immediately suggested a model for low pH-induced lipoprotein release, in which the propeller domain acts as an alternative intramolecular
Figure 3. Ribbon trace of the 3.7 A˚ resolution X-ray structure of the LDLR ectodomain determined at endosomal pH [57]. At low pH the central ligandbinding repeats contact the b-propeller domain, suggesting a model for low pH-induced lipoprotein release in which the propeller domain acts as an alternative intramolecular ligand for the ligand-binding domain at acidic pH. Modified, with permission, from Ref. [44] (www.molecule.org).
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ligand for the ligand-binding domain at acidic pH, displacing bound LDL [57,63,64]. Is the ability to form this long-range interface actually required to induce release of LDL at endosomal pH? Earlier studies showing that deletion of the entire EGFP region abrogates the release of ligands at low pH were certainly consistent with this model, but did not definitively implicate the b-propeller domain in release or directly evaluate the role of the long-range interface. Propeller domain deletion studies and domain swaps were thus performed to determine whether the YWTD propeller domain is required to trigger release of LDL at low pH. The results of these studies showed directly, and for the first time, that the propeller domain participates in mediating release of bound lipoproteins at endosomal pH [44]. What residues at the interface between the propeller and the LA repeats act as a pH sensor? Three interface histidines were particularly intriguing candidates [57] because of the ability of the histidine imidazole group to titrate between neutral and endosomal pH. H562 and H586 are on the surface of the b-propeller domain, pointing at LA5 and LA4, respectively, and H190 projects from the tip of a loop on LA5. Surprisingly, mutation of any individual histidine to tyrosine (mimicking the H190Y and H562Y FH mutations, and creating the additional H586Y mutant) did not interfere with the ability of the receptor to bind LDL at neutral pH on the cell surface, nor was the LDL-release activity of each mutant receptor diminished by more than w30% when compared with the native receptor. However, when all three histidines were
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simultaneously replaced by either alanine or tyrosine, the mutant receptors lost the ability to release bound LDL, showing that the histidine residues participate together in the induction of the long-range intramolecular interface to trigger ligand release at low pH [44]. What reversible interdomain movement(s) take place to interconvert the receptor between open (binding-active) and closed (binding-inactive) conformations? The unusual w90-degree angle in the interface between LA7 and EGF-A suggested the intriguing possibility that the linker between LA7 and EGF-A is a hinge, enabling the receptor to swing from an open conformation at neutral pH to a closed conformation at endosomal pH [64]. Indeed, a comparably short linker connecting LA modules 5 and 6 enables essentially unrestricted relative motion of these repeats, and the glycine residue in the linker between LA7 and EGF-A might confer enough flexibility to permit the hinge movement. Solution NMR studies of the isolated LA7-EGF-A domain pair, however, revealed an unanticipated finding: the linker connecting LA7 to EGF-A is not flexible and the interface between LA7 and EGF-A in the two-domain fragment is fixed and locked in virtually the same conformation at both neutral and endosomal pH. When the native interface between LA7 and EGF-A was disrupted by the introduction of two glycine mutations into the linker, in the context of the full-length receptor, the mutant receptor exhibited approximately half of the low-pH release activity of the native receptor [44], showing that the fixed orientation between LA7 and EGF-A is functionally important.
Figure 4. A schematic proposing how fixed and flexible connections among domains cooperate to permit interconversion between (a) open (neutral pH) and (b) closed (endosomal pH) conformations in response to pH. LA7, EGF-A and EGF-B constitute a rigid scaffold that is invariant with pH. Wavy lines identify modules linked by connections likely to be flexible at the indicated pH, with freedom of movement for the ligand-binding modules at neutral pH increasing as a function of distance from the rigid scaffold. LA modules are green, EGF-like modules are yellow and the b-propeller domain is pink. Reproduced, with permission, from Ref. [44] (www.molecule.org). www.sciencedirect.com
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Model for conformational rearrangements in the LDLR in response to pH The recently published structural and functional studies of the LDLR have led to a detailed model for the dynamic interdomain movements involved in its interconversion between the open and closed conformations (Figure 4). At neutral pH, the receptor samples an ensemble of open conformations, with the ligand-binding repeats moving essentially unrestricted relative to one another because they are connected by flexible linkers. This flexibility of the linkers enables the receptor to accommodate its shape to bind a heterogeneous mix of lipoprotein particles. When the receptor is exposed to low pH, it switches into the closed conformation, with the central ligand-binding repeats directly contacting the YWTD b-propeller domain of the EGFP region. To reorient at low pH, the receptor relies on intrinsic differences in the flexibility and rigidity of its intermodule connections. Three adjacent modules, LA7, EGF-A and EGF-B, situated between the LA4–5 modules and the propeller of the long-range interface, maintain
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fixed orientations with respect to one another throughout the physiologically relevant pH range [44,59,60]. The elbow formed by this trio of modules position the central ligand-binding repeats closer to the neighborhood of the propeller domain, essentially increasing the effective concentration of the b-propeller with respect to LA4 and LA5. The scaffold formed by LA7, EGF-A and EGF-B constrains the flanking mobile domains and limits the ligand-binding repeats to a smaller conformational search space, facilitating intramolecular closure at low pH. The flexibility of the linkers connecting adjacent ligandbinding repeats has a role in helping LA repeats 4 and 5 to reorient and dock onto the b-propeller, in addition to its role in facilitating ligand binding. The main unanswered question in LDLR structural biology at present is how the LA modules recognize lipoprotein ligands in the first place. Hints about the interfaces used by LA repeats in binding their partners come from the intramolecular interface in the LDLR [57] and the interface seen in the co-crystal structure of a
Figure 5. Comparison between the intramolecular LDLR interface and the VLDLR-virus interface (in stereo). (a) Intramolecular interface between the b-propeller domain and ligand-binding repeats LA4 and LA5 seen in the low pH crystal structure of the LDLR [57]. The propeller is blue, with interface side chains in cyan. LA4 is violet and LA5 is red, with side chains of the interface colored yellow. The calcium ions of the LA repeats are rendered as gray spheres. (b) Receptor-virus complex [65] illustrating contacts between two adjacent viral coat protein subunits and associated LA modules. Note the similarity in the recognition surface used by the LA modules of the LDLR and the LA modules bound to the virus. Figure prepared using the program Pymol (www.pymol.org). Reproduced, with permission, from Ref. [67] (www.nature.com). www.sciencedirect.com
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minor group human rhinovirus (HRV) coat protein and the third LA module of the VLDLR [65], which can serve as a rhinoviral receptor (Figure 5). In the low pH structure of the LDLR, conserved Ca2C-coordinating acidic residues of LA4 and LA5 form salt bridges with lysines K560 and K582, respectively, of the YWTD propeller domain. In the virus–receptor complex, similar ionic contacts are formed between a conserved lysine residue (K1224) of the viral coat protein VP1 and the analogous calcium-coordinating acidic residue of the LA module from the VLDLR. Indeed, K1224 is the only residue that is strictly conserved in all minor group HRVs and is required for viral infection, attesting to its importance in receptor recognition. Given that clusters of basic residues on apoE and apoB-100 are necessary for the high-affinity binding of lipoprotein particles to the LDLR, it is likely that lipoprotein recognition will also rely on ionic contacts between the calcium coordinating acidic residues and the apolipoproteins, as anticipated in the mid- and late-1980s [45,46]. Additional contacts between the LA domains and their partners might use the indole side chains of tryptophan residues that are frequently found in LA modules at the tip of a loop adjacent to the calcium coordination site.
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Concluding remarks Recent structural and functional studies of the LDLR have led to a detailed model for conformational rearrangements of the LDLR that drive release of bound lipoprotein particles at low pH. The data strongly suggest that exposure to low pH induces the formation of a long-range interface between two key ligand-binding modules and a b-propeller domain distal to them, converting the receptor from an open binding-active conformation to a closed binding-inactive conformation. Interconversion between open and closed conformations does not result from movement around a single pivot but rather occurs in the context of a pH-invariant scaffold serving as an anchor to restrict the conformational search space of the intramolecular partners. The elbow-shaped scaffold is combined with flexibility in the interdomain linkers between the ligand-binding repeats, and possibly in the connection between EGF-B and the propeller, to interconvert between an ensemble of open, binding-active conformations and the closed, low-pH conformation. Histidine residues in the low-pH intramolecular interface, presented by both the b-propeller and LA5, act in concert to ensure intramolecular closure at acidic pH. The question remains as to whether this delicate balance of interdomain mobility and rigidity will be a general feature shared by several of the interesting proteins that comprise the LDLR family.
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References 1 Goldstein, J.L. et al. (2001) Familial Hypercholesterolemia. In The Metabolic and Molecular Bases of Inherited Diseases (Scriver, C.S. et al., eds), pp. 2863–2913, McGraw Hill 2 Brown, M.S. and Goldstein, J.L. (1974) Familial hypercholesterolemia: defective binding of lipoproteins to cultured fibroblasts associated with impaired regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Proc. Natl. Acad. Sci. U. S. A. 71, 788–792 3 Goldstein, J.L. and Brown, M.S. (1973) Familial www.sciencedirect.com
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hypercholesterolemia: identification of a defect in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity associated with overproduction of cholesterol. Proc. Natl. Acad. Sci. U. S. A. 70, 2804–2808 Yamamoto, T. et al. (1984) The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA. Cell 39, 27–38 Russell, D.W. et al. (1984) Domain map of the LDL receptor: sequence homology with the epidermal growth factor precursor. Cell 37, 577–585 Russell, D.W. et al. (1989) Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins. J. Biol. Chem. 264, 21682–21688 Davis, C.G. et al. (1987) Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature 326, 760–765 Davis, C.G. et al. (1986) Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts. J. Biol. Chem. 261, 2828–2838 Lehrman, M.A. et al. (1985) Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain. Cell 41, 735–743 Davis, C.G. et al. (1987) The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis. J. Biol. Chem. 262, 4075–4082 Argraves, W.S. (2001) Members of the low density lipoprotein receptor family control diverse physiological processes. Front. Biosci. 6, D406–D416 May, P. and Herz, J. (2003) LDL receptor-related proteins in neurodevelopment. Traffic 4, 291–301 Schneider, W.J. and Nimpf, J. (2003) LDL receptor relatives at the crossroad of endocytosis and signaling. Cell. Mol. Life Sci. 60, 892–903 He, X. et al. (2004) LDL receptor-related proteins 5 and 6 in Wnt/betacatenin signaling: arrows point the way. Development 131, 1663–1677 Gent, J. and Braakman, I. (2004) Low-density lipoprotein receptor structure and folding. Cell. Mol. Life Sci. 61, 2461–2470 Jansens, A. et al. (2002) Coordinated nonvectorial folding in a newly synthesized multidomain protein. Science 298, 2401–2403 Bu, G. (2001) The roles of receptor-associated protein (RAP) as a molecular chaperone for members of the LDL receptor family. Int. Rev. Cytol. 209, 79–116 Li, Y. et al. (2002) Receptor-associated protein facilitates proper folding and maturation of the low-density lipoprotein receptor and its class 2 mutants. Biochemistry 41, 4921–4928 Culi, J. and Mann, R.S. (2003) Boca, an endoplasmic reticulum protein required for wingless signaling and trafficking of LDL receptor family members in Drosophila. Cell 112, 343–354 Hsieh, J.C. et al. (2003) Mesd encodes an LRP5/6 chaperone essential for specification of mouse embryonic polarity. Cell 112, 355–367 Culi, J. et al. (2004) Boca-dependent maturation of beta-propeller/ EGF modules in low-density lipoprotein receptor proteins. EMBO J. 23, 1372–1380 Innerarity, T.L. and Mahley, R.W. (1978) Enhanced binding by cultured human fibroblasts of apo-E-containing lipoproteins as compared with low density lipoproteins. Biochemistry 17, 1440–1447 Garcia, C.K. et al. (2001) Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein. Science 292, 1394–1398 He, G. et al. (2002) ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and AP-2. J. Biol. Chem. 277, 44044–44049 Brown, M.S. and Goldstein, J.L. (1986) A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34–47 Brown, M.S. et al. (1997) LDL-receptor structure. Calcium cages, acid baths and recycling receptors. Nature 388, 629–630 Wilson, D.J. et al. (1998) A World Wide Web site for low-density lipoprotein receptor gene mutations in familial hypercholesterolemia: sequence-based, tabular, and direct submission data handling. Am. J. Cardiol. 81, 1509–1511 Villeger, L. et al. (2002) The UMD-LDLR database: additions to the software and 490 new entries to the database. Hum. Mutat. 20, 81–87 Koivisto, U.M. et al. (2001) A novel cellular phenotype for familial hypercholesterolemia due to a defect in polarized targeting of LDL receptor. Cell 105, 575–585
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30 Simmons, T. et al. (1997) Human low density lipoprotein receptor fragment. Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli. J. Biol. Chem. 272, 25531–25536 31 Fisher, C. et al. (2004) A two-module region of the low-density lipoprotein receptor sufficient for formation of complexes with apolipoprotein E ligands. Biochemistry 43, 1037–1044 32 Daly, N.L. et al. (1995) Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor. Proc. Natl. Acad. Sci. U. S. A. 92, 6334–6338 33 Blacklow, S.C. and Kim, P.S. (1996) Protein folding and calcium binding defects arising from familial hypercholesterolemia mutations of the LDL receptor. Nat. Struct. Biol. 3, 758–762 34 Fass, D. et al. (1997) Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module. Nature 388, 691–693 35 Daly, N.L. et al. (1995) Three-dimensional structure of the second cysteine-rich repeat from the human low-density lipoprotein receptor. Biochemistry 34, 14474–14481 36 North, C.L. and Blacklow, S.C. (2000) Solution structure of the sixth LDL-A module of the LDL receptor. Biochemistry 39, 2564–2571 37 Dolmer, K. et al. (2000) NMR solution structure of complement-like repeat CR3 from the low density lipoprotein receptor-related protein. Evidence for specific binding to the receptor binding domain of human alpha(2)-macroglobulin. J. Biol. Chem. 275, 3264–3269 38 Simonovic, M. et al. (2001) Calcium coordination and pH dependence of the calcium affinity of ligand-binding repeat CR7 from the LRP. Comparison with related domains from the LRP and the LDL receptor. Biochemistry 40, 15127–15134 39 Huang, W. et al. (1999) NMR solution structure of complement-like repeat CR8 from the low density lipoprotein receptor-related protein. J. Biol. Chem. 274, 14130–14136 40 Wang, Q.Y. et al. (2002) Solution structure of the viral receptor domain of Tva and its implications in viral entry. J. Virol. 76, 2848–2856 41 Tonelli, M. et al. (2001) The solution structure of the viral binding domain of Tva, the cellular receptor for subgroup A avian leukosis and sarcoma virus. FEBS Lett. 509, 161–168 42 Kurniawan, N.D. et al. (2000) NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor. Protein Sci. 9, 1282–1293 43 North, C.L. and Blacklow, S.C. (1999) Structural independence of ligand-binding modules five and six of the LDL receptor. Biochemistry 38, 3926–3935 44 Beglova, N. et al. (2004) Cooperation between fixed and low pH-inducible interfaces controls lipoprotein release by the LDL receptor. Mol. Cell 16, 281–292 45 Sudhof, T.C. et al. (1985) The LDL receptor gene: a mosaic of exons shared with different proteins. Science 228, 815–822 46 Mahley, R.W. (1988) Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 240, 622–630 47 Innerarity, T.L. et al. (1979) Binding of arginine-rich (E) apoprotein after recombination with phospholipid vesicles to the low density lipoprotein receptors of fibroblasts. J. Biol. Chem. 254, 4186–4190 48 Wilson, C. et al. (1991) Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E. Science 252, 1817–1822
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49 Fisher, C.A. and Ryan, R.O. (1999) Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E. J. Lipid Res. 40, 93–99 50 Fisher, C.A. et al. (2000) The lipid-associated conformation of the low density lipoprotein receptor binding domain of human apolipoprotein E. J. Biol. Chem. 275, 33601–33606 51 Lund-Katz, S. et al. (2000) Effects of lipid interaction on the lysine microenvironments in apolipoprotein E. J. Biol. Chem. 275, 34459–34464 52 Lu, B. et al. (2000) Conformational reorganization of the four-helix bundle of human apolipoprotein E in binding to phospholipid. J. Biol. Chem. 275, 20775–20781 53 Segrest, J.P. et al. (2001) Structure of apolipoprotein B-100 in low density lipoproteins. J. Lipid Res. 42, 1346–1367 54 Orlova, E.V. et al. (1999) Three-dimensional structure of low density lipoproteins by electron cryomicroscopy. Proc. Natl. Acad. Sci. U. S. A. 96, 8420–8425 55 Lunin, V.Y. et al. (2001) Low-resolution data analysis for low-density lipoprotein particle. Acta Crystallogr. D Biol. Crystallogr. 57, 108–121 56 Beglova, N. et al. (2001) Backbone dynamics of a module pair from the ligand-binding domain of the LDL receptor. Biochemistry 40, 2808–2815 57 Rudenko, G. et al. (2002) Structure of the LDL receptor extracellular domain at endosomal pH. Science 298, 2353–2358 58 Boswell, E.J. et al. (2004) Global defects in the expression and function of the low density lipoprotein receptor (LDLR) associated with two familial hypercholesterolemia mutations resulting in misfolding of the LDLR epidermal growth factor-AB pair. J. Biol. Chem. 279, 30611–30621 59 Kurniawan, N.D. et al. (2001) NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor. J. Mol. Biol. 311, 341–356 60 Saha, S. et al. (2001) Solution structure of the LDL receptor EGF-AB pair: a paradigm for the assembly of tandem calcium binding EGF domains. Structure (Camb.) 9, 451–456 61 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 62 Jeon, H. et al. (2001) Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair. Nat. Struct. Biol. 8, 499–504 63 Innerarity, T.L. (2002) Structural biology. LDL receptor’s betapropeller displaces LDL. Science 298, 2337–2339 64 Rudenko, G. and Deisenhofer, J. (2003) The low-density lipoprotein receptor: ligands, debates and lore. Curr. Opin. Struct. Biol. 13, 683–689 65 Verdaguer, N. et al. (2004) X-ray structure of a minor group human rhinovirus bound to a fragment of its cellular receptor protein. Nat. Struct. Mol. Biol. 11, 429–434 66 Fisher, C. et al. (2001) Familial Hypercholesterolaemia. In Encyclopedia of Life Sciences. Macmillan Publisher, Nature Publishing Group 67 Blacklow, S.C. (2004) Catching the common cold. Nat. Struct. Mol. Biol. 11, 388–390
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The LDL receptor: how acid pulls the trigger Natalia Beglova and Stephen C. Blacklow Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
The low-density lipoprotein receptor normally carries lipoprotein particles into cells, and releases them upon delivery to the low pH milieu of the endosome. Recent structural and functional studies of the receptor, combined with the plethora of prior knowledge about normal receptor function and the effects of diseaseassociated mutations that cause familial hypercholesterolemia, reveal a detailed molecular model for how the acidic environment of the endosome triggers the release of bound lipoprotein particles. Remarkably, the receptor dynamically interconverts between open (ligand-active) and closed (ligand-inactive) conformations in response to pH, relying on a specific arrangement of fixed and flexible interdomain connections to facilitate efficient binding and release of its lipoprotein ligands. Introduction The low-density lipoprotein receptor (LDLR) is responsible for transporting cholesterol-containing lipoprotein particles from the circulation into cells. Patients with lossof-function mutations in the LDLR gene have familial hypercholesterolemia (FH), an autosomal dominant genetic disorder affecting w1 in 500 individuals worldwide. In heterozygotes, FH is characterized clinically by elevated concentrations of plasma low-density lipoprotein (LDL) and cholesterol, and an increased risk of atherosclerosis and coronary heart disease. Untreated patients with homozygous FH have dramatically elevated levels of
plasma LDL and cholesterol, and typically die from heart disease at an early age [1]. The LDLR was first discovered by Brown and Goldstein in their search for the molecular basis underlying FH [2,3]. They found that fibroblasts from normal individuals bind LDL with high affinity and specificity, and suppress endogenous cholesterol synthesis when cultured in the presence of LDL. By contrast, cultured fibroblasts from homozygous FH patients did not suppress endogenous cholesterol synthesis and failed to take up LDL-cholesterol from the culture media. The gene encoding the LDLR was cloned and sequenced about ten years later [4,5] (GenBank accession number AH002776). Domain organization of the LDLR The mature LDLR is a modular, single-pass transmembrane glycoprotein of 839 amino acids (Figure 1). The seven adjacent LDL receptor type-A (LA) modules at the N-terminal end of the receptor are responsible for binding to lipoproteins [6]. Immediately C-terminal to these ligand-binding repeats is a region with homology to the epidermal growth factor precursor (EGFP), which consists of two epidermal growth factor-like (EGF) modules, followed by a YWTD domain and a third EGF module. This part of the LDLR is implicated in the release of bound lipoproteins at low pH [7]. Between the EGFP and the membrane is a region rich in serine and threonine residues that undergoes O-linked glycosylation. The
Key: LA repeat EGF-like module β-propeller domain O-linked sugar region NPxY endocytosis motif YxxL endocytosis motif
LDLR VLDLR ApoER2
LRP1 Megalin LRP5/6 Ti BS
Figure 1. Proteins of the LDLR family. The domain organization of the core family members is illustrated schematically. Corresponding author: Blacklow, S.C. ([email protected]). Available online 11 April 2005 www.sciencedirect.com 0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.03.007
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glycosylation region is believed to be a spacer separating the LDLR functional domains from the cell surface because a mutated LDLR lacking this spacer sequence is indistinguishable from the native receptor with respect to ligand binding, internalization and receptor recycling [8]. The O-linked sugar domain is then followed by a transmembrane segment and a 50-residue cytoplasmic tail, which is required for receptor localization in clathrincoated pits and for receptor endocytosis [9,10].
Cellular itinerary of the LDLR The pathway traversed by individual LDLR molecules in the cell is schematically illustrated in Figure 2. Immediately after synthesis, the precursor of the mature receptor migrates with an apparent molecular weight of 120 kDa on SDS–PAGE. Upon transport to the Golgi, the LDLR undergoes extensive O-linked glycosylation into the mature 160 kDa form found at the cell surface. Folding of the LDLR occurs in the ER in a non-vectorial manner [15,16]. Early in the folding process, the N-terminal cysteine-rich repeats form non-native disulfide bonds that reshuffle into native disulfide pairs, with the first LA repeat (LA1) acquiring its native fold late in the folding process [16]. The receptor associated protein (RAP), a common chaperone of LDLR family proteins, binds to the N-terminal LA repeats, facilitating their proper folding, www.sciencedirect.com
(3)
*
*
* *
*
*
Clathrincoated pit
(4)
*
*
Coated vesticles
*
*
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LDLR family It is now evident that the LDLR constitutes the founding member of an entire class of transmembrane receptors (Figure 1). Members of the LDLR family conduct a wide range of physiologic functions, both as transporters of lipoprotein particles and other structurally unrelated ligands, and also as receptors or co-receptors in signal transduction cascades (see [11–14] for recent reviews). Although many proteins with LA, EGF or YWTD extracellular modules exist in the protein sequence database, each core member of the LDLR contains all three signature structural units of the LDLR. The most closely related family members are the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), which share w50% homology to the LDLR and to each other, and have an almost identical domain organization. These receptors, which are expressed in the brain, serve as receptors for a large secreted signaling protein called Reelin and have a crucial role in the migration of cortical neurons during development. Two giants of the family, LRP and megalin, have myriad and complex roles as both scavenger transporters and signaling molecules. Together, they constitute a second subgroup, in which the LA, EGF and YWTD modules are still arranged in a similar pattern. LRP-5 and LRP-6 still contain all three types of modules; however, the organization is different, with the YWTD domains preceding a series of three LA repeats adjacent to the membrane spanning domain. LRP-5 and LRP-6, and their homologues in frogs and flies, are co-receptors for Wnt signaling during development. Other distantly related receptors, with functions that are less welldefined, include LRP3, LRP9 and LRP12, which have LA and CUB domains in their extracellular portion, and SorLA-1 (LRP11).
LDL
LDLR
Recycling vesicle
(5)
*
Endosome H+
*
(2)
* Lysosome Amino acids
*
*
Cholesterol
(1) ER
Ti BS
Figure 2. Cellular itinerary of the LDLR. (1) Receptors are first synthesized by ribosomes and folded in the endoplasmic reticulum. (2) Next, the receptors are glycosylated in the Golgi and transported to the cell surface. (3) At the plasma membrane, receptors bind lipoprotein ligands. (4) Internalization occurs via clathrin-coated pits, which ultimately deliver receptor–ligand complexes to endosomes. (5) After the bound lipoproteins are released, the receptors recycle back to the cell surface. Each of these 5 steps is also associated with a corresponding class of LDLR mutations found in FH. Initially adapted from Ref. [25], and modified, with permission, from Ref. [66] (www.els.net).
and preventing the premature binding of co-expressed ligands [17,18]. A second kind of chaperone protein, called Boca in Drosophila and Mesd in the mouse [19,20], is believed to assist folding of the YWTD b-propeller domains of LDLR family proteins [21]. Binding of circulating lipoproteins to the LDLR occurs at the cell surface. The most important physiological ligand for the LDLR is LDL, which contains a single copy of the apolipoprotein B-100 (apoB-100) as its primary protein component; 65–70% of plasma cholesterol in humans circulates in the form of LDL. In addition, the receptor also exhibits high-affinity binding of lipoproteins that contain multiple copies of apolipoprotein E (apoE), like b-migrating forms of very lowdensity lipoprotein (b-VLDL) and certain intermediate density lipoproteins [2,22]. Receptor-ligand complexes enter cells by endocytosis via clathrin-coated pits. Endocytosis of the LDLR requires the presence of an NPXY sequence in the cytoplasmic tail. Hobbs, Cohen and co-workers recently identified a gene that was mutated in an autosomal
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recessive form of FH [23]. The gene, which they called ARH-1, encodes a protein with a PTB domain capable of binding the NPXY sequence of the LDLR, a canonical clathrin-binding sequence LLDLE, and a sequence recognized by the b2-adaptin subunit of AP-2, the other major structural component of clathrin-coated pits [24]. These findings strongly suggest that ARH-1 serves as an adaptor protein that links the LDLR to the endocytic machinery by simultaneously binding to the NPXY sequence of the receptor cytoplasmic tail, clathrin and the AP-2 adaptor. After receptor–ligand complexes are taken up into clathrin-coated pits, they are then delivered to endosomes, where the low pH environment triggers release of the bound lipoprotein particles. Subsequently, the receptors return to the cell surface in a process called receptor recycling. By contrast, the released lipoprotein particles proceed to lysosomes, where the cholesterol esters are hydrolyzed to free cholesterol [25]. Remarkably, each LDLR cycles through its itinerary continuously, regardless of whether or not it is carrying bound cargo, completing each passage in 10–30 min. With a half-life of 16–24 h, a typical LDLR molecule recycles through 100 ‘acid baths’ or more before being degraded [26]. In patients with FH, more than 1000 mutations in the LDLR have been identified that interfere with one or more of these central events required for normal receptor function [27,28]. The mutations have been grouped into five classes, depending on what kind of receptor defect is observed (Figure 2). Thus, class 1 mutants fail to produce detectable amounts of protein; class 2 mutants have a partial or complete transport defect; class 3 mutants are impaired in ligand binding; class 4 mutants fail to localize in clathrincoated pits and are internalization-defective; and class 5 mutants exhibit a ligand release and recycling defect [1]. Mellman’s group recently showed that the G823D FH mutation, which lies toward the C-terminal end of the cytoplasmic tail beyond the NPXY sequence, interferes with proper sorting of the LDLR to the basolateral membrane of polarized epithelial cells, identifying yet another, sixth class of mutation implicated in FH [29].
Structure–function correlates in the LDLR ligand-binding domain Early mutational studies probing structure–function relationships in the LDLR showed that the LA repeats constitute the ligand-binding domain of the receptor [6]. Deletion of any repeat from LA3 to LA7, in addition to deletion of the first EGF repeat, interferes with the cell-surface binding of LDL. However, a recombinant form of the ligand-binding domain, including only the seven ligand-binding repeats, refolded after expression in bacteria, retains the ability to bind LDL, showing that the rest of the protein is not necessary for LDL binding once the protein is no longer on the cell surface [30]. Constraints on b-VLDL binding are less stringent because only deletion of LA5 effectively abrogates b-VLDL binding. Indeed, recent in vitro binding assays have identified the LA4-LA5 fragment of the LDLR as sufficient for binding apoE–DMPC complexes, which www.sciencedirect.com
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are believed to mimic b-VLDL with regard to receptor binding [31]. A picture of how the receptor recognizes lipoprotein ligands first began to emerge from structural studies of individual ligand-binding modules and module pairs, in combination with structural and biochemical studies of the receptor-binding domain of apoE. The NMR solution structure of the first ligand-binding repeat of the LDLR revealed a novel fold with three disulfide bonds and little secondary structure [32]. Studies investigating the requirements for formation of the native disulfide bonds of LA5 showed that calcium ions were required for proper folding, and that the bound calcium ion was required to maintain the structural integrity of the module [33]. Subsequently, the X-ray structure of LA5 identified four highly conserved acidic residues near the C-terminal end of the module, which stabilize the structure by participating in the coordination of a single bound calcium ion in an octahedral ‘cage’ [34]. The FH mutations in LA5 disrupt module folding by altering either the calcium coordinating residues or other key scaffolding residues in the module. In the absence of calcium, the structural integrity of the LA modules is lost and, as a result, the receptor fails to bind lipoprotein particles. NMR and crystallographic studies of other LA repeats, either individually (LA1, LA2, LA6, CR3, CR7, CR8 and TVA; reviewed in [32]; [35–41]), or in the context of module pairs, LA1–2, LA5–6 or LA7-EGF-A [42–44], also confirm the structural importance of calcium in specifying the LA module fold and are fully consistent with this interpretation of the role of calcium in LDL and b-VLDL binding. Another notable consequence of calcium coordination by the LA modules is that the conserved C-terminal acidic residues become clustered on one face of the module, creating a discrete patch of electronegative surface potential on each LA module. Both apoB and apoE, the protein moieties of LDL and b-VLDL, respectively, have a series of conserved basic residues that are required for binding to the LDLR, leading to the proposal that ligand recognition by the LDLR relies on electrostatic complementarity between receptor (acidic) and ligand (basic) [45,46]. One reason that it has been difficult to address this proposal at atomic resolution is that the receptor-binding domain of apoE requires association with lipid to become a high-affinity ligand for the LDLR [47]. Although the details of lipid activation are incompletely understood, the receptor-binding domain of apoE clearly changes its conformation upon lipid association. Before lipid activation, the receptor binding domain of apoE adopts an elongated four-helix bundle structure [48], whereas association with lipid causes the bundle to open up so that the hydrophobic interiors of the helices pack against the aliphatic tails of the lipids [49–52]. In the lipid-free form of apoE, the basic region required for binding to the LDLR lies on the solvent-exposed face of the fourth helix of the bundle, potentially in position to form contacts with the calcium-coordinating acidic clusters of the LA repeats. Why lipid activation is needed to enhance affinity, therefore, remains a puzzle. Perhaps, lipid association enhances the affinity for the receptor by inducing a specific curvature of the helices responsible for receptor
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recognition, by creating a multivalent ligand, by creating a composite protein-lipid surface required for recognition, or by some combination of these mechanisms. Role of flexible linkers connecting the LA repeats in facilitating ligand binding An unusual feature of the natural ligands for the LDLR is that they are highly heterogeneous in size and shape, posing a distinct challenge for recognition by a single receptor. Although LDL particles typically contain only a single copy ˚ in of apoB-100, they range in size from w180–270 A diameter [53–55]. b-VLDL particles have multiple copies of apoE per particle and are even larger, up to ˚ in diameter. 400 A How does the LDLR adjust its shape to bind different lipoprotein particles of varying size and composition? One important feature contributing to the versatility of the receptor in binding different ligands is that the linkers connecting adjacent ligand-binding modules seem to be flexible, even though all intermodule linkers, with the exception of the 12-residue linker connecting LA4 to LA5, are only 4–5 residues long. Studies of the LA1–LA2 and LA5–LA6 module pairs by solution NMR show that the linkers connecting the modules enable essentially unrestricted movement of one module with respect to the next in each case [42,56]. More generally, the absence of contacts between adjacent ligand-binding modules in the X-ray structure of the LDLR ectodomain (see section entitled Central role of the b-propeller in the intramolecular interactions at low pH) suggests that the rest of the ligand-binding modules are also unconstrained with respect to one another [57]. This intrinsic flexibility of the linkers connecting the ligand-binding modules, enabling relative freedom of motion for adjacent LA modules, permits the LDLR to accommodate its shape to bind a variety of heterogeneous lipoprotein particles. Structure and function of the EGFP domain The region of the LDLR that is implicated in release of bound lipoproteins at low pH is the epidermal growth factor precursor homology domain (EGFP). This part of the receptor lies immediately C-terminal to the ligandbinding domain and consists of two EGF-like modules (EGF-A and EGF-B), followed by a YWTD b-propeller domain and a third EGF-like module (EGF-C). When the entire EGFP region is deleted, the receptor is capable of binding b-VLDL and, to a lesser extent, LDL, but fails to release it at low pH [7,44,58]. The mutant receptor also fails to recycle efficiently and is degraded more rapidly than the native receptor after ligand binding [7]. The frequent occurrence of FH mutations in the EGFP region of the receptor also attests to its functional importance. Structural studies show that, in contrast to the LA modules of the ligand-binding domain, which are connected to one another by flexible linkers, some of the interdomain orientations of adjacent modules in the EGFP are fixed. The EGF-AB domain pair, solved independently by two different groups by solution NMR, adopts a rigid elongated conformation at neutral pH, with the interdomain orientation defined by calcium coordination at a site between the domains and by hydrophobic interdomain www.sciencedirect.com
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packing interactions [59,60]. The crystal structure of a receptor fragment containing the YWTD b-propeller (the fold of which was first predicted by Springer [61]) and its two flanking EGF modules, solved at neutral pH, revealed that the C-terminal EGF module (EGF-C) packs tightly against the b-propeller, whereas the EGF module preceding the propeller domain (EGF-B) is disordered in the crystal [62]. Elucidation of the structure enabled rationalization of the effects of many of the FH mutations located in the YWTD-EGF-C domain pair: one class of mutations alters side chains that form conserved packing and hydrogen-bonding interactions in the interior and between propeller blades, whereas a second subset of FH mutations are located at the interface between the propeller and EGF-C module, suggesting a structural requirement for maintaining the integrity of the interdomain interface. Although the X-ray structure provided important atomic details about interdomain relationships among modules within the EGFP region, and also about FH mutations, it did not immediately explain why the EGFP is required to promote the release of bound lipoproteins upon exposure to acidic pH.
Central role of the b-propeller in intramolecular interactions at low pH A key breakthrough that provided a structural rationale for the role of the EGFP domain in low-pH induced release of bound lipoproteins came when Deisenhofer’s group, in collaboration with Brown and Goldstein, determined the ˚ resolution crystal structure of the LDLR ectodomain to 3.7 A at endosomal pH [57]. In the low-pH structure, the receptor adopts a closed conformation, with a long-range intramolecular interface between the top face of the YWTD b-propeller and two of the central ligand-binding repeats, LA4 and LA5 (Figure 3). The closed structure immediately suggested a model for low pH-induced lipoprotein release, in which the propeller domain acts as an alternative intramolecular
Figure 3. Ribbon trace of the 3.7 A˚ resolution X-ray structure of the LDLR ectodomain determined at endosomal pH [57]. At low pH the central ligandbinding repeats contact the b-propeller domain, suggesting a model for low pH-induced lipoprotein release in which the propeller domain acts as an alternative intramolecular ligand for the ligand-binding domain at acidic pH. Modified, with permission, from Ref. [44] (www.molecule.org).
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ligand for the ligand-binding domain at acidic pH, displacing bound LDL [57,63,64]. Is the ability to form this long-range interface actually required to induce release of LDL at endosomal pH? Earlier studies showing that deletion of the entire EGFP region abrogates the release of ligands at low pH were certainly consistent with this model, but did not definitively implicate the b-propeller domain in release or directly evaluate the role of the long-range interface. Propeller domain deletion studies and domain swaps were thus performed to determine whether the YWTD propeller domain is required to trigger release of LDL at low pH. The results of these studies showed directly, and for the first time, that the propeller domain participates in mediating release of bound lipoproteins at endosomal pH [44]. What residues at the interface between the propeller and the LA repeats act as a pH sensor? Three interface histidines were particularly intriguing candidates [57] because of the ability of the histidine imidazole group to titrate between neutral and endosomal pH. H562 and H586 are on the surface of the b-propeller domain, pointing at LA5 and LA4, respectively, and H190 projects from the tip of a loop on LA5. Surprisingly, mutation of any individual histidine to tyrosine (mimicking the H190Y and H562Y FH mutations, and creating the additional H586Y mutant) did not interfere with the ability of the receptor to bind LDL at neutral pH on the cell surface, nor was the LDL-release activity of each mutant receptor diminished by more than w30% when compared with the native receptor. However, when all three histidines were
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simultaneously replaced by either alanine or tyrosine, the mutant receptors lost the ability to release bound LDL, showing that the histidine residues participate together in the induction of the long-range intramolecular interface to trigger ligand release at low pH [44]. What reversible interdomain movement(s) take place to interconvert the receptor between open (binding-active) and closed (binding-inactive) conformations? The unusual w90-degree angle in the interface between LA7 and EGF-A suggested the intriguing possibility that the linker between LA7 and EGF-A is a hinge, enabling the receptor to swing from an open conformation at neutral pH to a closed conformation at endosomal pH [64]. Indeed, a comparably short linker connecting LA modules 5 and 6 enables essentially unrestricted relative motion of these repeats, and the glycine residue in the linker between LA7 and EGF-A might confer enough flexibility to permit the hinge movement. Solution NMR studies of the isolated LA7-EGF-A domain pair, however, revealed an unanticipated finding: the linker connecting LA7 to EGF-A is not flexible and the interface between LA7 and EGF-A in the two-domain fragment is fixed and locked in virtually the same conformation at both neutral and endosomal pH. When the native interface between LA7 and EGF-A was disrupted by the introduction of two glycine mutations into the linker, in the context of the full-length receptor, the mutant receptor exhibited approximately half of the low-pH release activity of the native receptor [44], showing that the fixed orientation between LA7 and EGF-A is functionally important.
Figure 4. A schematic proposing how fixed and flexible connections among domains cooperate to permit interconversion between (a) open (neutral pH) and (b) closed (endosomal pH) conformations in response to pH. LA7, EGF-A and EGF-B constitute a rigid scaffold that is invariant with pH. Wavy lines identify modules linked by connections likely to be flexible at the indicated pH, with freedom of movement for the ligand-binding modules at neutral pH increasing as a function of distance from the rigid scaffold. LA modules are green, EGF-like modules are yellow and the b-propeller domain is pink. Reproduced, with permission, from Ref. [44] (www.molecule.org). www.sciencedirect.com
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Model for conformational rearrangements in the LDLR in response to pH The recently published structural and functional studies of the LDLR have led to a detailed model for the dynamic interdomain movements involved in its interconversion between the open and closed conformations (Figure 4). At neutral pH, the receptor samples an ensemble of open conformations, with the ligand-binding repeats moving essentially unrestricted relative to one another because they are connected by flexible linkers. This flexibility of the linkers enables the receptor to accommodate its shape to bind a heterogeneous mix of lipoprotein particles. When the receptor is exposed to low pH, it switches into the closed conformation, with the central ligand-binding repeats directly contacting the YWTD b-propeller domain of the EGFP region. To reorient at low pH, the receptor relies on intrinsic differences in the flexibility and rigidity of its intermodule connections. Three adjacent modules, LA7, EGF-A and EGF-B, situated between the LA4–5 modules and the propeller of the long-range interface, maintain
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fixed orientations with respect to one another throughout the physiologically relevant pH range [44,59,60]. The elbow formed by this trio of modules position the central ligand-binding repeats closer to the neighborhood of the propeller domain, essentially increasing the effective concentration of the b-propeller with respect to LA4 and LA5. The scaffold formed by LA7, EGF-A and EGF-B constrains the flanking mobile domains and limits the ligand-binding repeats to a smaller conformational search space, facilitating intramolecular closure at low pH. The flexibility of the linkers connecting adjacent ligandbinding repeats has a role in helping LA repeats 4 and 5 to reorient and dock onto the b-propeller, in addition to its role in facilitating ligand binding. The main unanswered question in LDLR structural biology at present is how the LA modules recognize lipoprotein ligands in the first place. Hints about the interfaces used by LA repeats in binding their partners come from the intramolecular interface in the LDLR [57] and the interface seen in the co-crystal structure of a
Figure 5. Comparison between the intramolecular LDLR interface and the VLDLR-virus interface (in stereo). (a) Intramolecular interface between the b-propeller domain and ligand-binding repeats LA4 and LA5 seen in the low pH crystal structure of the LDLR [57]. The propeller is blue, with interface side chains in cyan. LA4 is violet and LA5 is red, with side chains of the interface colored yellow. The calcium ions of the LA repeats are rendered as gray spheres. (b) Receptor-virus complex [65] illustrating contacts between two adjacent viral coat protein subunits and associated LA modules. Note the similarity in the recognition surface used by the LA modules of the LDLR and the LA modules bound to the virus. Figure prepared using the program Pymol (www.pymol.org). Reproduced, with permission, from Ref. [67] (www.nature.com). www.sciencedirect.com
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minor group human rhinovirus (HRV) coat protein and the third LA module of the VLDLR [65], which can serve as a rhinoviral receptor (Figure 5). In the low pH structure of the LDLR, conserved Ca2C-coordinating acidic residues of LA4 and LA5 form salt bridges with lysines K560 and K582, respectively, of the YWTD propeller domain. In the virus–receptor complex, similar ionic contacts are formed between a conserved lysine residue (K1224) of the viral coat protein VP1 and the analogous calcium-coordinating acidic residue of the LA module from the VLDLR. Indeed, K1224 is the only residue that is strictly conserved in all minor group HRVs and is required for viral infection, attesting to its importance in receptor recognition. Given that clusters of basic residues on apoE and apoB-100 are necessary for the high-affinity binding of lipoprotein particles to the LDLR, it is likely that lipoprotein recognition will also rely on ionic contacts between the calcium coordinating acidic residues and the apolipoproteins, as anticipated in the mid- and late-1980s [45,46]. Additional contacts between the LA domains and their partners might use the indole side chains of tryptophan residues that are frequently found in LA modules at the tip of a loop adjacent to the calcium coordination site.
4 5 6
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Concluding remarks Recent structural and functional studies of the LDLR have led to a detailed model for conformational rearrangements of the LDLR that drive release of bound lipoprotein particles at low pH. The data strongly suggest that exposure to low pH induces the formation of a long-range interface between two key ligand-binding modules and a b-propeller domain distal to them, converting the receptor from an open binding-active conformation to a closed binding-inactive conformation. Interconversion between open and closed conformations does not result from movement around a single pivot but rather occurs in the context of a pH-invariant scaffold serving as an anchor to restrict the conformational search space of the intramolecular partners. The elbow-shaped scaffold is combined with flexibility in the interdomain linkers between the ligand-binding repeats, and possibly in the connection between EGF-B and the propeller, to interconvert between an ensemble of open, binding-active conformations and the closed, low-pH conformation. Histidine residues in the low-pH intramolecular interface, presented by both the b-propeller and LA5, act in concert to ensure intramolecular closure at acidic pH. The question remains as to whether this delicate balance of interdomain mobility and rigidity will be a general feature shared by several of the interesting proteins that comprise the LDLR family.
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