683

The low-density lipoprotein receptor: ligands, debates and lore

Gabby Rudenko and Johann Deisenhofery
Like pieces belonging to a large mosaic, the structures of low-density lipoprotein receptor (LDL-R) modules have been elucidated one by one in recent years. LDL-Rs localized on hepatocytes play an important role in removing cholesterol-transporting LDL particles from the plasma by receptor-mediated endocytosis. Key steps in this process involve the LDL-R binding LDL at neutral pH at the cell surface and, after internalization, releasing it again at acidic pH in the endosomes. How the modules of the LDL-R might interact within the intact receptor to carry out ligand binding and release has been revealed by the recent crystal structure of the extracellular domain of the LDL-R.
Addresses Department of Biochemistry, yHoward Hughes Medical Institute, UT Southwestern Medical Center, 5323 Harry Hines Boulevard Y 4-206, Dallas, TX 75390-9050, USA y e-mail: Johann.Deisenhofer@UTSouthwestern.edu

on the cell surface at neutral pH (i.e. in the plasma environment). After internalization, the receptor releases LDL in the endosomes at acidic pH, enabling lysosomal degradation of LDL and recycling of the receptor. Ligand binding by the LDL-R requires Ca2. The LDL-R, by being able to bind and release LDL, plays a crucial role in cholesterol metabolism. Efcient clearance of LDL via LDL-R is important because more than a thousand LDLR mutations are known to lead to familial hypercholesterolemia (FH), a disease characterized by increased levels of plasma LDL, early onset coronary heart disease and atherosclerosis [2,3,4]. The extracellular domain of LDL-R can be subdivided into a ligand-binding domain containing seven cysteinerich repeats, and an epidermal growth factor (EGF)precursor homology domain consisting of two EGF-like repeats and a b propeller followed by a third EGF-like repeat (Figure 1). The modules in the ligand-binding domain associate with different ligands to varying extents, whereas the EGF-precursor homology domain is essential for ligand release induced by acidic pH, although modules in this domain are probably also involved in binding some ligands [5,6]. Over the past few years, structures of the different LDLR modules have been solved either alone or as tandem arrays. In 2002, a large portion of the extracellular domain of LDL-R was solved by X-ray crystallography, giving the rst insight into the arrangement of the modules as a virtually complete entity. In this review, we summarize recent structural information obtained for LDL-R and discuss its impact on two main issues of debate: how LDL-R binds and releases ligand.

Current Opinion in Structural Biology 2003, 13:683689 This review comes from a themed issue on Proteins Edited by Christian Cambillau and David I Stuart 0959-440X/$ see front matter 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2003.10.001

Abbreviations cbEGF calcium-binding EGF EGF epidermal growth factor FH familial hypercholesterolemia Kd dissociation constant LDL low-density lipoprotein LDL-R LDL receptor LRP LDL-R-related protein PDB Protein Data Bank VLDL very low density lipoprotein

Revealing the structure of the LDL-R

Ligand-binding domain

Introduction
In order to use cholesterol, our bodies must overcome two challenges: transporting the water-insoluble molecule through the blood and preventing its accumulation. The transport problem is resolved by packaging esteried cholesterol together with apolipoproteins in large, soluble lipoprotein particles (including low-density lipoprotein [LDL], intermediate-density lipoprotein [IDL] and very low density lipoprotein [VLDL] particles) [1]. The removal of excess plasma cholesterol traveling in the form of LDL is accomplished by outtting hepatocytes with LDL receptors (LDL-Rs) [2]. The LDL-R binds LDL
www.current-opinion.com

The rst modules of LDL-R to be structurally elucidated were cysteine-rich repeats from the ligand-binding domain: R1 (by NMR [7]), R2 (by NMR [8]), R5 (by X-ray crystallography [9]) and R6 (by NMR [10,11]). Each cysteine-rich repeat consists of roughly 40 residues that form two loops (or lobes) lacking regular secondary structure (a helices or b strands), which are held together by disulde bonds (Figure 2a). The C-terminal loop houses a cluster of acidic residues that form a Ca2-binding site, which was rst shown in R5 by crystallography [9]. Modules in tandem repeats do not interact with each other (Figure 2b), as shown by the structures of R1R2 [12], R5 R6* (where R6* contains the mutation Met243Leu) [13] and R5R6 [14]. Different biophysical techniques have given Kd values in the nanomolar to micromolar range for
Current Opinion in Structural Biology 2003, 13:683689

684 Proteins

Figure 1

N terminus R1 R2 R3 R4 R5 R6 R7 A Ligand-binding domain

B propeller C CD

C terminus

EGF-precursor homology domain

TMS

Current Opinion in Structural Biology

Schematic representation of human LDL-R. The LDL-R is made up of an extracellular domain (containing the ligand-binding and EGF-precursor homology domains), a single transmembrane segment (TMS) and a small cytoplasmic domain (CD). The ligand-binding domain comprises seven cysteine-rich repeats (R1R7) connected by short linkers of 45 residues, with the exception of the linker between R4 and R5, which is 12 residues long. The EGF-precursor homology domain contains three EGF-like repeats (AC) and a b propeller. The N and C termini are indicated.

Ca2 at neutral pH, indicating that these modules have high-afnity Ca2-binding sites (Table 1).
EGF-precursor homology domain

The EGF-precursor homology domain contains three EGF-like repeats: the tandem pair AB and C (Figure 1). The EGF-like repeats comprise 4050 residues, which form two pairs of short antiparallel b strands (Figure 2c). In solution, the AB tandem forms a rigid rod (Figure 2d) [15,16]. The C-terminal b sheet in module B is not well dened, showing motion on a nanosecond timescale, which led Saha et al. [15] and Kurniawan et al. [16] to suggest that this region becomes stabilized upon interaction with the b propeller of the EGF-precursor homology domain. The C module does pack against the b propeller [17,18] (Figure 2f). Modules A and B contain Ca2-binding sites with low micromolar Kd values

(Table 1), whereas module C does not bind Ca2 [1517]. The orientation of A with respect to B seems to be heavily inuenced by Ca2 binding in the B site; NMR relaxation data indicate that Ca2 rigidies the linker residues between the last cysteine of A and the rst cysteine of B residues that are also involved in chelating Ca2 [15,16]. The EGF-like repeats thus resemble the cysteine-rich repeats in being small, disulde-containing modules that can contain Ca2-binding sites. The last module to be solved, the b propeller, contains six blades, each made up of a b sheet with four antiparallel b strands (predicted rst [19] and later shown by crystallography [17]) (Figure 2e). A characteristic Tyr-TrpThr-Asp (YWTD) or YWTD-like consensus sequence repeats every 40 residues, for a total of six times, and is located on strand 2 of each blade (Figure 2e).

Table 1 Dissociation constants for Ca2R of modules in the LDL-R extracellular domain. Module Kd for Ca2 pH References repeats) [35] [36] [13] [22] [22] [37] [13] [13,37] [13] [38] [24] [24] Referred to by Bieri et al. [38] Trp fluorescence; Tb3 0.4 mM; Gd3 0.3 mM Referred to by Bieri et al. [38] Fluorescence Trp fluorescence Isothermal titration calorimetry; experiment not suited to measure Kd < 100 nM Isothermal titration calorimetry As R5R6 pair; Trp fluorescence As R5R6* pair, R6* contains M243L mutation; Trp fluorescence Tyr fluorescence R6* contains M243L mutation; Tyr fluorescence Averaged over two sites; HummelDreyer zonal chromatography EC50 measurement (i.e. [Ca2]free producing 50% of maximal Trp fluorescence response) EC50 measurement (i.e. [Ca2]free producing 50% of maximal Trp fluorescence response) Comments

Ligand-binding domain (cysteine-rich R1 10 mM ? R1 7 mM 7.4 R2 14 mM ? R5 70 nM 7.0 R5 36 nM 7.0 R5 0.5 mM 7.4 R5 13.1 mM 5.0 R5 170 nM 7.0 R5 44 nM 7.0 R6 200 nM 7.0 R6* 203 nM 7.0 R1R2 48 mM 7.5 (R1R7) A 5060 nM 7.4 (R1R7) A 10 mM 5.5

EGF-precursor homology domain (EGF-like repeats) A 45 mM 7.4 [27] As AB tandem; 2D NMR (Thr294 and Tyr315); technique not suited to measure Kd 100 mM B 1020 mM 7.5 [27] As AB tandem; value inferred from indirect measurements AB 7.2 mM 7.5 [27] As AB tandem; averaged over two sites (site B dominates); Tyr fluorescence AB 22 mM 7.5 [27] As AB tandem; averaged over two sites (site B dominates); measured with BAPTA

Current Opinion in Structural Biology 2003, 13:683689

www.current-opinion.com

Structural insights into LDL receptor functioning Rudenko and Deisenhofer 685

Figure 2

Figure 3

(a) N

(b) C C R2 R1 R7 C N (d) N C A B A C B propeller

Current Opinion in Structural Biology

R5 R6 R4 R3 R2

(c) N

(e)

(f)

propeller

Current Opinion in Structural Biology

Modules found in the extracellular domain of the LDL-R. Disulfide bonds are shown in white and yellow ball-and-stick representation, red spheres indicate Ca2 ions. (a) Crystal structure of cysteine-rich repeat R5 ([9], PDB code 1AJJ). The two lobes are held together by three disulfide bonds with connectivity Cys1Cys3, Cys2Cys5 and Cys4Cys6. Binding of Ca2 engages the consensus sequence Xn(mainchain carbonyl)XXAsp/Asnn3(sidechain)XXn5(mainchain carbonyl)X Asp/Asn/Glun7(sidechain)XXXXXAspn13(carboxylate)Asp/ Glun14(carboxylate). (b) NMR structure of the tandem repeat R1R2 ([12], PDB code 1F5Y). A four-residue linker tethers the N-terminal cysteine-rich repeat R1 (dark blue) to the C-terminal cysteine-rich repeat R2 (light blue) with considerable flexibility. R1 and R2 essentially behave independently of each other. (c) NMR structure of EGF-like repeat A, as seen in the AB tandem ([15], PDB code 1HJ7). Other structures of A are known, alone [27] or in tandem with B [16]. The module contains three disulfide bonds with connectivity Cys1Cys3, Cys2Cys4 and Cys5Cys6. (d) NMR structure of EGF-like repeats A and B in a tandem pair ([15], PDB code 1HJ7). C-terminal module B has a canonical Ca2-binding site made up of two stretches of consensus amino acids: stretch 1, Asp/Asn/GluIle/ValAsp/Asn/GluGlu/Asp/ GlnCys1; and stretch 2, Cys3XAsp*/Asn*XXXXTyr/PheXCys4, where the asterisk indicates an optionally hydroxylated residue [32]. N-terminal module A has a noncanonical Ca2-binding site, as stretch 1 has a glycine residue at the first position instead of an acidic residue [15,27]. (e) Crystal structure of the b propeller as seen in a fragment with C ([17], PDB code 1IJQ). Strands 14 are labeled in magenta for one of the blades. Each strand 2 contains a YWTD(-like) consensus sequence. The YWTD repeats are similar to the WD40 repeats found in seven-bladed b propellers, except that, in the latter, consensus tryptophan and aspartic acid residues are located on strand 3, although they do fulfill the same structural role. (f) Crystal structure of a fragment containing the b propeller and C ([17], PDB code 1IJQ). The EGF-like repeat C (dark blue) packs against the side of the b propeller. Although C contains three disulfide bonds, no Ca2 is observed. Figures made with Molscript [33] and Raster3D [34].

Crystal structure of the extracellular domain of LDL-R at acidic pH ([18], PDB code 1N7D). R1 is not visible, probably because of flexibility. Modules are labeled according to the nomenclature in Figure 1. Disulfide bonds are shown in white and yellow ball-and-stick representation, Ca2 are shown as red spheres and a segment of poor density in R3 for mainchain atoms is represented by a broken line. N-linked carbohydrates visible in the structure have been omitted for clarity.

Extracellular domain

How do seven cysteine-rich repeats, three EGF-like repeats and the b propeller interact with each other in the context of the complete extracellular domain? A glimpse of these interactions has been recently provided by a crystal structure containing R1R7, AB, the b propeller and C, solved at acidic pH 5.3 [18]. The modules form a long chain that doubles back on itself so that the ligand-binding domain arches over the EGFprecursor homology domain (Figure 3). In the ligandbinding domain, most of the cysteine-rich repeats are independent of each other and contacted only by linker residues; the exceptions are R4 and R5 (see below). By contrast, the EGF-precursor homology domain seems to be a rigid entity with interfaces between A and B, B and the b propeller, and the b propeller packed against C. Unlike in the solution structure, the C-terminal two b strands of B are well ordered and packed against the b propeller. Although A and B form a linear array, the two modules have rotated relative to each other by 408 around the rod axis, compared with their orientation in the isolated tandem array in solution. Indirect evidence suggests that the Ca2-binding sites in the cysteine-rich repeats and in EGF-like repeats A and B are occupied at pH 5.3 [18]. Astonishingly, R4 and R5 dock independently side by side on the b propeller; this nding is signicant because R4 and R5 are known to be very important for ligand binding. The interface formed between R4, R5 and the b
Current Opinion in Structural Biology 2003, 13:683689

The tryptophan and aspartic acid in the consensus sequence provide important stabilizing interactions within each blade as well as between blades.
www.current-opinion.com

686 Proteins

propeller involves the Ca2-binding loops of the two cysteine-rich repeats, three histidines (His190, His562 and His586) and four tryptophans (Trp144, Trp193, Trp515 and Trp541). Most of the residues found at this interface are identical or highly conserved across 15 sequences of the LDL-R and the related VLDL receptor (VLDL-R). Thus, on the basis of the crystal structure at acidic pH, it appears that a exible ligand-binding domain contacts a rigid EGF-precursor homology domain through very specic interactions between R4, R5 and the b propeller.

Translating structural knowledge of LDL-R into biochemical understanding

How does LDL-R bind different lipoproteins?

The basis of LDL binding by LDL-R is thought to reside in a conserved stretch(es) of basic residues shared by apolipoprotein B and apolipoprotein E, and exposed on the surface of LDL and VLDL particles. These basic residues have long been thought to interact with clusters of conserved acidic residues located on each of the cysteine-rich repeats in the ligand-binding domain of LDL-R [1,20,21]. Doubt was cast, however, when many of these acidic residues were found to contribute to Ca2binding sites [9]. But not all of the negative charges in the clusters are obscured upon cation binding (e.g. Glu237 in R6 [11] and Asp27 in cysteine-rich repeat CR7 of the LDL-R-related protein [LRP] [22]) and Ca2 association could serve to x chelating and/or surrounding residues in a competent mode for lipoprotein binding. Although the exact mechanism of ligand association remains to be determined, it seems clear that LDL-R is ideally suited to recognize lipoprotein particles by concatenating a series of recognition domains that can ex with respect to each other and wrap around particles of varying size and with variable epitopes.
How does LDL-R release bound ligands upon arrival in the endosome?

that acidic pH too causes the dissociation of Ca2 in a fragment of R1R7 and the A repeat, with subsequent ligand loss. However, NMR spectra monitoring the Ca2chelating residues Asp26, Asp36 and Glu37 in R1 in the presence of Ca2 show no chemical shift changes for these residues in the pH range 3.96.8, indicating that these residues remain chelated to Ca2 [25]. Admittedly, the situation is not straightforward. Studies on LDL-R and related proteins have shown that different cysteinerich repeats can bind Ca2 at neutral pH with similar afnity, but show different proles of Ca2 dissociation as a function of decreasing pH: compare R5 in LDL-R, and CR3, CR7 and CR8 in LRP [22] (Tables 1 and 2). By contrast, and defying prediction, Ca2-binding sites formed by identical ligands can show different Ca2 afnities as well as characteristic Ca2-afnity proles as a function of pH [22]. In the case of calcium-binding EGF (cbEGF)-like repeats, things are even more complicated because repeats can behave cooperatively and Ca2 binding then depends on the surrounding modules: compare cbEGF13 alone and in tandem, and cbEGF32 alone and in tandem (Table 2). The controversial issue seems to be whether a decrease in Ca2-binding afnity concomitant with arrival in the endosomes would be large enough to explain complete ligand release by the LDL-R. It seems to come down to the balance between the concentration of Ca2 in the endosomes (estimated to be around 10 mM [26,27]), the afnity of each module for Ca2 at acidic pH (initially with ligand still bound!) and the importance of that particular module for ligand binding.
Potential role of histidines in ligand release

As yet, pH-dependent sidechain conformations that might explain ligand release by LDL-R have not been identied. NMR studies of modules in the ligand-binding domain have been carried out in the pH range 3.97.5, with no mention of conformations related to ligand release. Furthermore, a detailed comparison of sidechain conformations in crystal structures of the b propeller and C repeat solved at pH 7.5 [17], and the ectodomain solved at pH 5.3 [18] is not warranted because of the limited resolution (3.7 A) of the latter structure.
Potential role of Ca2 in ligand release

Could histidines play a role in pH-regulated ligand release? In other proteins, histidine residues have been implicated in regulating ligand release upon endocytosis, either directly by affecting ligand binding or indirectly by causing large conformational changes [2830]. A cluster of three histidines is found at the R4, R5 and b propeller interface. Two of these histidines are mutated in FH patients (His190Tyr and His562Tyr [3,4]), although the effects of these mutations on protein function have not been studied in vitro. The close proximity of the histidines to each other, their general location between the two negatively charged Ca2-binding sites and their sequence conservation invite further study into their role in ligand release.
Potential role of domain rearrangements in ligand release

Could a decrease in Ca2-binding afnity as a function of pH explain the disruption of ligand binding? Certainly, articially removing Ca2 from LDL-R (e.g. with EDTA) completely prevents ligand binding [23]. Also, on the basis of tryptophan uorescence studies and 45 Ca2 blots, Dirlam-Schatz and Attie [24] suggested
Current Opinion in Structural Biology 2003, 13:683689

Could major domain rearrangements take place as LDL-R cycles between neutral and acidic pH, thereby prompting ligand release? The crystal structure of the ectodomain at acidic pH shows interactions between modules not seen before in structures of fragments [18]. Firstly, A swivels with respect to B. Secondly, B packs extensively against the b propeller an interaction
www.current-opinion.com

Structural insights into LDL receptor functioning Rudenko and Deisenhofer 687

Table 2 Dissociation constants for Ca2R of modules in proteins related to LDL-R. Module Kd for Ca2 pH References [35]y [22] [22] [22] [22] [35]y [22] [22] [39] [40] [39] [40] [41] [42] [42] Comments* Trp fluorescence; shows small decrease in Kd as a function of acidic pH (<CR8) Isothermal titration calorimetry Isothermal titration calorimetry Isothermal titration calorimetry Isothermal titration calorimetry Trp fluorescence; shows large decrease in Kd as a function of acidic pH (>CR3) Isothermal titration calorimetry Isothermal titration calorimetry As tandem cbEGF12cbEGF13; NMR As tandem cbEGF12cbEGF13; As tandem cbEGF13cbEGF14; NMR (monitoring Tyr2149) As tandem cbEGF32cbEGF33; As tandem cbEGF32cbEGF33; 2D NMR (monitoring Phe1093 in cbEGF12) 2D NMR (monitoring Tyr1136 in cbEGF13) NMR 2D NMR (monitoring Tyr2149 in cbEGF32) 2D NMR (monitoring Phe2188 in cbEGF33)

Cysteine-rich repeats found in LRP CR3 24 mM 7.4 CR3 8.0 mM 7.4 CR3 12.5 mM 5.0 CR7 12.6 mM 7.4 CR7 640 mM 5.0 CR8 13 mM 7.4 CR8 6.1 mM 7.4 CR8 20.5 mM 5.0 EGF-like repeats found in human fibrillin-1 cbEGF12 1.6 mM 6.5 cbEGF13 23 mM 6.5 cbEGF13 27 mM 6.5 cbEGF14 100 mM 6.5 cbEGF32 4 mM 7.4 cbEGF32 9.2 mM 6.5 cbEGF33 0.35 mM 6.5

Kd values for Ca2 are given for isolated modules except where stated in the Comments column that the module is part of a tandem repeat or a fragment. yReferences [22,35] report conflicting trends for CR3 and CR8 as a function of decreasing pH.

that does not take place at neutral pH in a fragment containing B, the b propeller and C (only the b propeller and C are ordered and thus visible in the latter crystal structure) [17]. The buried surface between B and the b propeller seems to be important because ve FH mutations map to this area; all ve mutations alter the charge balance at the surface, although two probably primarily cause protein misfolding. The third, most striking, interFigure 4

action between modules is the extensive interface formed by R4 and R5 with the b propeller. Deletion mutagenesis studies have indicated the importance of R4 and R5 for ligand binding [5], so it is signicant that these modules bury what must be ligand-binding surfaces against the b propeller (obscuring them) at acidic pH in the crystal structure. Recently, albeit in another biochemical system, a similar structural mode of interaction between a b

(a)

(b)

G293

LDL

LDL G375

Extracellular pH

Endosomal pH
Current Opinion in Structural Biology

Model showing how the LDL-R might open and close to bind ligand as a function of pH. (a) At neutral pH on the cell surface, the LDL-R may present itself as an elongated molecule, extending ligand-binding epitopes into solution. (b) At acidic pH (<6.0) in the endosomes, the LDL-R may undergo a conformational change, whereby the b propeller of the EGF-precursor homology domain binds to the ligand-binding domain, displacing bound ligand. Black arrows indicate speculative hinge regions between R7 of the ligand-binding domain and A of the EGF-precursor homology domain (Gly293), and between B and the b propeller (Gly375). The extracellular domain is colored as in Figure 3; LDL, the ligand of LDL-R, is shown as a light-green disk. www.current-opinion.com Current Opinion in Structural Biology 2003, 13:683689

688 Proteins

propeller and cysteine-rich-like modules has been observed between the nidogen-1 GIII b propeller and its ligand, the laminin fragment LE3-5 [31]. Taking these results together, the conformational rearrangement of LDL-R modules in space seems likely to accompany ligand binding and release.
Potential special role of the b-propeller domain in ligand release

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Segrest JP, Jones MK, De Loof H, Dashti N: Structure of apolipoprotein B-100 in low density lipoproteins. J Lipid Res 2001, 42:1346-1367.

It is tantalizing to speculate that the b propeller provides an intramolecular mechanism for displacing lipoprotein particles. In this model (Figure 4), at neutral pH the LDL-R would extend itself in an elongated form to bind ligand, but on exposure to acidic pH it would fold back on itself as the b propeller competed with ligand for binding to the ligand-binding domain. Putative hinge points allowing this movement are located between the R7 and A repeats (Gly293), and the B repeat and the b propeller (Gly375); in fact, the FH mutation Gly375Ser maps to the latter position. Gel ltration studies have indicated that the extracellular domain of LDL-R indeed has an extended shape at neutral pH, but is signicantly more compact at acidic pH [18]. In addition, the fragment R1R4 does not associate with the rest of the extracellular domain at neutral pH, but it does at acidic pH [18], supporting the idea of pH-dependent conformations. Further biochemical and biophysical studies are clearly needed to verify such a mechanism of intramolecular displacement. Perhaps revisiting experiments from many years ago provides the most clues. Studies in which full-length LDL-R and an LDL-R truncated to just the ligandbinding domain were expressed on the cell surface showed that, on its own, the ligand-binding domain, while still able to bind LDL, could no longer release ligand at acidic pH (either upon endocytosis or articially at the cell surface), unlike the full-length receptor [6]. Because at neutral pH, 10 mM suramin (which binds LDL) could release LDL equally well from both the truncated ligand-binding domain and the full-length receptor, Davis et al. [6] concluded that components in the EGF-precursor homology domain were specically needed for acid-triggered ligand release in the endosomes. These results undermine the idea that, at endosomal pH, ligand release is dominated by a decrease in Ca2 afnity, but they are compatible with an intramolecular displacement mechanism involving the bpropeller domain.

Goldstein JL, Hobbs HH, Brown MS: Familial hypercholesterolemia. In The Metabolic & Molecular Bases of Inherited Disease, vol 2. Edited by Scriver CR et al. New York: McGraw-Hill; 2001:2863-2913. An up-to-date review detailing genetic and biochemical aspects of LDLR, and its relevance to the disease FH. 3. 4. The low-density lipoprotein receptor (LDL-R) gene in FH on World Wide Web URL: http://www.ucl.ac.uk/fh ` Villeger L, Abifadel M, Allard D, Rabes JP, Thiart R, Kotze MJ, Beroud C, Junien C, Boileau C, Varret M: The UMD-LDLR database: additions to the software and 490 new entries to the database. Hum Mutat 2002, 20:81-87. Russell DW, Brown MS, Goldstein JL: Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins. J Biol Chem 1989, 264:21682-21688. Davis CG, Goldstein JL, Sudhof TC, Anderson RG, Russell DW, Brown MS: Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature 1987, 326:760-765. Daly NL, Scanlon MJ, Djordjevic JT, Kroon PA, Smith R: Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor. Proc Natl Acad Sci USA 1995, 92:6334-6338. Daly NL, Djordjevic JT, Kroon PA, Smith R: Three-dimensional structure of the second cysteine-rich repeat from the human low-density lipoprotein receptor. Biochemistry 1995, 34:14474-14481. Fass D, Blacklow S, Kim PS, Berger JM: Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module. Nature 1997, 388:691-693.

2. 

5.

6.

7.

8.

9.

10. Clayton D, Brereton IM, Kroon PA, Smith R: Three-dimensional NMR structure of the sixth ligand-binding module of the human LDL receptor: comparison of two adjacent modules with different ligand binding specicities. FEBS Lett 2000, 479:118-122. 11. North CL, Blacklow SC: Solution structure of the sixth LDL-A module of the LDL receptor. Biochemistry 2000, 39:2564-2571. 12. Kurniawan ND, Atkins AR, Bieri S, Brown CJ, Brereton IM, Kroon PA, Smith R: NMR structure of a concatemer of the rst and second ligand-binding modules of the human low-density lipoprotein receptor. Protein Sci 2000, 9:1282-1293. 13. North CL, Blacklow SC: Structural independence of ligandbinding modules ve and six of the LDL receptor. Biochemistry 1999, 38:3926-3935. 14. Beglova N, North CL, Blacklow SC: Backbone dynamics of a module pair from the ligand-binding domain of the LDL receptor. Biochemistry 2001, 40:2808-2815. 15. Saha S, Boyd J, Werner JM, Knott V, Handford PA, Campbell ID,  Downing AK: Solution structure of the LDL receptor EGF-AB pair: a paradigm for the assembly of tandem calcium binding EGF domains. Structure 2001, 9:451-456. The authors of these two papers [15,16] used NMR to show that, unlike adjacent cysteine-rich repeats in the ligand-binding domain, the tandem pair of EGF-like repeats A and B in the EGF-precursor homology domain interact with each other to form a rod. 16. Kurniawan ND, Aliabadizadeh K, Brereton IM, Kroon PA, Smith R:  NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor. J Mol Biol 2001, 311:341-356. See annotation to [15]. www.current-opinion.com

Conclusions
The recent structural studies described in this review have shaped our thoughts on how LDL-R may bind and release ligands. Hopefully, this wealth of structural information will lead to the design of targeted studies using biophysical and mutagenesis techniques to conrm our thinking on LDL-R function.
Current Opinion in Structural Biology 2003, 13:683689

Structural insights into LDL receptor functioning Rudenko and Deisenhofer 689

17. Jeon H, Meng W, Takagi J, Eck MJ, Springer TA, Blacklow SC:  Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTDEGF domain pair. Nat Struct Biol 2001, 8:499-504. Crystallographic study revealing the structure of a fragment containing the b propeller and the EGF-like repeat C at neutral Ph. 18. Rudenko G, Henry L, Henderson K, Ichtchenko K, Brown MS,  Goldstein JL, Deisenhofer J: Structure of the LDL receptor extracellular domain at endosomal pH. Science 2002, 298:2353-2358. The crystal structure of the almost complete extracellular domain of LDLR at acidic pH. 19. Springer TA: An extracellular b-propeller module predicted in lipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor precursor, and extracellular matrix components. J Mol Biol 1998, 283:837-862. 20. Brown MS, Deuel TF, Basu SK, Goldstein JL: Inhibition of the binding of low-density lipoprotein to its cell surface receptor in human broblasts by positively charged proteins. J Supramol Struct 1978, 8:223-234. 21. Innerarity TL, Weisgraber KH, Rall SC Jr, Mahley RW: Functional domains of apolipoprotein E and apolipoprotein B. Acta Med Scand Suppl 1987, 715:51-59. 22. Simonovic M, Dolmer K, Huang W, Strickland DK, Volz K,  Gettins PG: Calcium coordination and pH dependence of the calcium afnity of ligand binding repeat CR7 from LRP. Comparison with related domains from the LRP and the LDL receptor. Biochemistry 2001, 40:15127-15134. An informative study aimed at understanding the pH dependence of Ca2 binding to cysteine-rich repeats of LDL-R and a related protein, LRP. 23. Schneider WJ, Beisiegel U, Goldstein JL, Brown MS: Purication of the low density lipoprotein receptor, an acidic glycoprotein of 164 000 molecular weight. J Biol Chem 1982, 257:2664-2673. 24. Dirlam-Schatz KA, Attie AD: Calcium induces a conformational change in the ligand binding domain of the low density lipoprotein receptor. J Lipid Res 1998, 39:402-411. 25. Atkins AR, Brereton IM, Kroon PA, Lee HT, Smith R: Calcium is essential for the structural integrity of the cysteine-rich, ligand binding repeat of the low-density lipoprotein receptor. Biochemistry 1998, 37:1662-1670. 26. Gerasimenko JV, Tepikin AV, Petersen OH, Gerasimenko OV: Calcium uptake via endocytosis with rapid release from acidifying endosomes. Curr Biol 1998, 8:1335-1338. 27. Malby S, Pickering R, Saha S, Smallridge R, Linse S, Downing AK:  The rst epidermal growth factor-like domain of the lowdensity lipoprotein receptor contains a noncanonical calcium binding site. Biochemistry 2001, 40:2555-2563. An informative study aimed at understanding the pH dependence of Ca2 binding to EGF-like repeats A and B of LDL-R. 28. Doi T, Kurasawa M, Higashino K-I, Imanishi T, Mori T, Naito M, Takahashi K, Kawabe Y, Wada Y, Matsumoto A et al.: The histidine interruption of an a-helical coiled coil allosterically mediates a pH-dependent ligand dissociation from macrophage scavenger receptors. J Biol Chem 1994, 269:25598-25604.

29. Wragg S, Drickamer K: Identication of amino acid residues that determine pH dependence of ligand binding to the asialoglycoprotein receptor during endocytosis. J Biol Chem 1999, 274:35400-35406. 30. Garrett TPJ, McKern NM, Lou M, Elleman TC, Adams TE, Lovrecz GO, Zhu H-J, Walker F, Frenkel MJ, Hoyne PA et al.: Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor a. Cell 2002, 110:763-773. 31. Takagi J, Yang Y, Liu J-H, Wang J-H, Springer TA: Complex between nidogen and laminin fragments reveals a paradigmatic b-propeller interface. Nature 2003, 424:969-974. 32. Rand MD, Lindblom A, Carlson J, Villoutreix BO, Steno J: Calcium binding to tandem repeats of EGF-like modules. Expression and characterization of the EGF-like modules of human Notch1 implicated in receptorligand interactions. Protein Sci 1997, 6:2059-2071. 33. Kraulis PJ: MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 1991, 24:946-950. 34. Merritt EA, Bacon DJ: Raster3D: photorealistic molecular graphics. Methods Enzymol 1997, 277:505-524. 35. Dolmer K, Huang W, Gettins PG: Characterization of the calcium site in two complement-like domains from the low-density lipoprotein receptor-related protein (LRP) and comparison with a repeat from the low-density lipoprotein receptor. Biochemistry 1998, 37:17016-17023. 36. Blacklow SC, Kim PS: Protein folding and calcium binding defects arising from familial hypercholesterolemia mutations of the LDL receptor. Nat Struct Biol 1996, 3:758-762. 37. North CL, Blacklow SC: Evidence that familial hypercholesterolemia mutations of the LDL receptor cause limited local misfolding in an LDL-A module pair. Biochemistry 2000, 39:13127-13135. 38. Bieri S, Atkins AR, Lee HT, Winzor DJ, Smith R, Kroon PA: Folding, calcium binding, and structural characterization of a concatemer of the rst and second ligand-binding modules of the low-density lipoprotein receptor. Biochemistry 1998, 37:10994-11002. 39. Smallridge RS, Whiteman P, Doering K, Handford PA, Downing KA: EGF-like domain calcium afnity modulated by N-terminal domain linkage in human brillin-1. J Mol Biol 1999, 286:661-668. 40. Whiteman P, Downing AK, Smallridge R, Winship PR, Handford PA: A Gly!Ser change causes defective folding in vitro of calciumbinding epidermal growth factor-like domains from factor IX and brillin-1. J Biol Chem 1998, 273:7807-7813. 41. Handford P, Downing AK, Rao Z, Hewett DR, Sykes BC, Kielty CM: The calcium binding properties and molecular organization of epidermal growth factor-like domains in human brillin-1. J Biol Chem 1995, 270:6751-6756. 42. Knott V, Downing KA, Cardy CM, Handford P: Calcium binding properties of an epidermal growth factor-like domain pair from human brillin-1. J Mol Biol 1996, 255:22-27.

www.current-opinion.com

Current Opinion in Structural Biology 2003, 13:683689