the Low-Density in Receptor

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The low-density lipoprotein receptor: ligands, debates and loreGabby Rudenko and Johann Deisenhofery

Like pieces belonging to a large mosaic, the structures oflow-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.

AddressesDepartment of Biochemistry, yHoward Hughes Medical Institute,UT Southwestern Medical Center, 5323 Harry Hines Boulevard

Y 4-206, Dallas, TX 75390-9050, USAye-mail: [email protected]

Current Opinion in Structural Biology 2003, 13:683689

This review comes from a themed issue on

ProteinsEdited 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 EGFEGF epidermal growth factor

FH familial hypercholesterolemia

Kd dissociation constantLDL low-density lipoproteinLDL-R LDL receptorLRP LDL-R-related protein

PDB Protein Data BankVLDL very low density lipoprotein

IntroductionIn order to use cholesterol, our bodies must overcome twochallenges: transporting the water-insoluble moleculethrough the blood and preventing its accumulation.The transport problem is resolved by packaging esterifiedcholesterol together with apolipoproteins in large, solublelipoprotein particles (including low-density lipoprotein[LDL], intermediate-density lipoprotein [IDL] and verylow density lipoprotein [VLDL] particles) [1]. Theremoval of excess plasma cholesterol traveling in the formof LDL is accomplished by outfitting hepatocytes withLDL receptors (LDL-Rs) [2]. The LDL-R binds LDL

on the cell surface at neutral pH (i.e. in the plasmaenvironment). After internalization, the receptor releasesLDL in the endosomes at acidic pH, enabling lysosomaldegradation of LDL and recycling of the receptor. Ligandbinding by the LDL-R requires Ca2. The LDL-R, bybeing able to bind and release LDL, plays a crucial role incholesterol metabolism. Efficient clearance of LDL viaLDL-R is important because more than a thousand LDL-R mutations are known to lead to familial hypercholes-terolemia (FH), a disease characterized by increasedlevels of plasma LDL, early onset coronary heart diseaseand atherosclerosis [2,3,4].

The extracellular domain of LDL-R can be subdividedinto a ligand-binding domain containing seven cysteine-rich repeats, and an epidermal growth factor (EGF)-precursor homology domain consisting of two EGF-likerepeats and a b propeller followed by a third EGF-likerepeat (Figure 1). The modules in the ligand-bindingdomain associate with different ligands to varyingextents, whereas the EGF-precursor homology domainis essential for ligand release induced by acidic pH,although modules in this domain are probably alsoinvolved in binding some ligands [5,6].

Over the past few years, structures of the different LDL-R modules have been solved either alone or as tandem

arrays. In 2002, a large portion of the extracellular domainof LDL-R was solved by X-ray crystallography, giving thefirst insight into the arrangement of the modules as avirtually complete entity. In this review, we summarizerecent structural information obtained for LDL-R anddiscuss its impact on two main issues of debate: howLDL-R binds and releases ligand.

Revealing the structure of the LDL-RLigand-binding domain

The first modules of LDL-R to be structurally elucidatedwere cysteine-rich repeats from the ligand-bindingdomain: R1 (by NMR [7]), R2 (by NMR [8]), R5 (by

X-ray crystallography [9]) and R6 (by NMR [10,11]). Eachcysteine-rich repeat consists of roughly 40 residues thatform two loops (or lobes) lacking regular secondary struc-ture (a helices or b strands), which are held together bydisulfide bonds (Figure 2a). The C-terminal loop houses acluster of acidic residues that form a Ca2-binding site,which was first shown in R5 by crystallography [9]. Mod-ules in tandem repeats do not interact with each other(Figure 2b), as shown by the structures of R1R2 [12], R5R6* (where R6* contains the mutation Met243Leu) [13]and R5R6 [14]. Different biophysical techniques havegiven Kd values in the nanomolar to micromolar range for

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Ca2 at neutral pH, indicating that these modules havehigh-affinity 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 res-idues, 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 defined, showing motion on a nanosecondtimescale, which led Saha et al. [15] and Kurniawan et al.

[16] to suggest that this region becomes stabilized uponinteraction 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 seemsto be heavily influenced by Ca2 binding in the B site;NMR relaxation data indicate that Ca2 rigidifies thelinker residues between the last cysteine of A and the firstcysteine of B residues that are also involved in chelat-ing Ca2 [15,16]. The EGF-like repeats thus resemble

the cysteine-rich repeats in being small, disulfide-con-taining 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 first [19] and later shown by crystal-lography [17]) (Figure 2e). A characteristic Tyr-Trp-

Thr-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).

Figure 1

Current Opinion in Structural Biology

R1 R2 R3 R4 R5 R6 R7 A B propeller C C terminusN terminus

Ligand-binding

domain TMS

CDEGF-precursor homology

domain

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 residueslong. The EGF-precursor homology domain contains three EGF-like repeats (AC) and a b propeller. The N and C termini are indicated.

Table 1

Dissociation constants for Ca2R of modules in the LDL-R extracellular domain.

Module Kd for Ca2 pH References Comment s

Ligand-binding domain (cysteine-rich repeats)

R1 10 mM ? Referred to by Bieri et al. [38]

R1 7 mM 7.4 [35] Trp fluorescence; Tb3 0.4 mM; Gd3 0.3 mM

R2 14 mM ? Referred to by Bieri et al. [38]

R5 70 nM 7.0 [36] Fluorescence

R5 36 nM 7.0 [13] Trp fluorescence

R5 0.5 mM 7.4 [22] Isothermal titration calorimetry; experiment not suited to measure Kd < 100 nM

R5 13.1 mM 5.0 [22] Isothermal titration calorimetry

R5 170 nM 7.0 [37] As R5R6 pair; Trp fluorescenceR5 44 nM 7.0 [13] As R5R6* pair, R6* contains M243L mutation; Trp fluorescence

R6 200 nM 7.0 [13,37] Tyr fluorescence

R6* 203 nM 7.0 [13] R6* contains M243L mutation; Tyr fluorescence

R1R2 48 mM 7.5 [38] Averaged over two sites; HummelDreyer zonal chromatography

(R1R7) A 5060 nM 7.4 [24] EC50 measurement (i.e. [Ca2]free producing 50% of maximal Trp fluorescence response)

(R1R7) A 10 mM 5.5 [24] EC50 measurement (i.e. [Ca2]free producing 50% of maximal Trp fluorescence response)

EGF-precursor homology domain (EGF-like repeats)

A 45mM 7.4 [27] As AB tandem;2D NMR(Thr294and Tyr315); technique notsuitedto measure Kd100 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

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The tryptophan and aspartic acid in the consensus

sequence provide important stabilizing interactions

within each blade as well as between blades.

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 bpropeller 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 EGF-

precursor homology domain (Figure 3). In the ligand-

binding 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). Bycontrast, 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 twomodules 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 sug-

gests 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 finding is significant becauseR4 and R5 are known to be very important for ligand

binding. The interface formed between R4, R5 and the b

Figure 2


(a) (b)

(c) (d)

(e) (f)

propeller

NN

A B

C

N

C

C

C

N

C

R2 R1

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 Ca 2 is

observed. Figures made with Molscript [33] and Raster3D [34].

Figure 3


propeller

R5

R6

R7

R4

R3

R2

CB

A

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.

Structural insights into LDL receptor functioning Rudenko and Deisenhofer 685



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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 flexible ligand-bindingdomain contacts a rigid EGF-precursor homology domain

through very specific interactions between R4, R5 and theb propeller.

Translating structural knowledge of LDL-Rinto biochemical understandingHow does LDL-R bind different lipoproteins?

The basis of LDL binding by LDL-R is thought to residein 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 Ca2-

binding 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 associa-

tion could serve to fix chelating and/or surrounding res-idues 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 byconcatenating a series of recognition domains that can

flex with respect to each other and wrap around particlesof varying size and with variable epitopes.

How does LDL-R release bound ligands upon arrival in

the endosome?

As yet, pH-dependent sidechain conformations that

might explain ligand release by LDL-R have not been

identified. NMR studies of modules in the ligand-bindingdomain 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 andC 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 a decrease in Ca2-binding affinity as a function ofpH explain the disruption of ligand binding? Certainly,

artificially removing Ca2 from LDL-R (e.g. withEDTA) completely prevents ligand binding [23]. Also,

on the basis of tryptophan fluorescence studies and45Ca2 blots, Dirlam-Schatz and Attie [24] suggested

that acidic pH too causes the dissociation of Ca2 in a

fragment of R1R7 and the A repeat, with subsequentligand loss. However, NMR spectra monitoring the Ca2-

chelating 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 thatthese residues remain chelated to Ca2 [25]. Admittedly,the situation is not straightforward. Studies on LDL-R

and related proteins have shown that different cysteine-

rich repeats can bind Ca2 at neutral pH with similar

affinity, but show different profiles of Ca2 dissociationas a function of decreasing pH: compare R5 in LDL-R,

and CR3, CR7 and CR8 inLRP [22] (Tables 1 and 2).By

contrast, and defying prediction, Ca2-binding sites

formed by identical ligands can show different Ca2

affinities as well as characteristic Ca2-affinity profilesas 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 affinity concomitant with arrivalin 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 inthe endosomes (estimated to be around 10 mM [26,27]),

the affinity of each module for Ca2 at acidic pH (initiallywith ligand still bound!) and the importance of that

particular module for ligand binding.

Potential role of histidines 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 ofthree histidines is found at the R4, R5 and b propellerinterface. 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 histi-

dines to each other, their general location between the

two negatively charged Ca2

-binding sites and theirsequence conservation invite further study into their role

in ligand release.

Potential role of domain rearrangements in ligand release

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 theb propeller an interaction

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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 bpropeller seems to be important because five FH muta-tions map to this area; all five mutations alter the chargebalance at the surface, although two probably primarily

cause protein misfolding. The third, most striking, inter-

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 significant that these modulesbury what must be ligand-binding surfaces against the b

propeller (obscuring them) at acidic pH in the crystalstructure. Recently, albeit in another biochemical system,

a similar structural mode of interaction between a b

Table 2

Dissociation constants for Ca2R of modules in proteins related to LDL-R.

Module Kd for Ca2 pH References Comments*

Cysteine-rich repeats found in LRP

CR3 24 mM 7.4 [35]y Trp fluorescence; shows small decrease in Kd as a function of acidic pH (CR3)

CR8 6.1 mM 7.4 [22] Isothermal titration calorimetry

CR8 20.5 mM 5.0 [22] Isothermal titration calorimetry

EGF-like repeats found in human fibrillin-1

cbEGF12 1.6 mM 6.5 [39] As tandem cbEGF12cbEGF13; 2D NMR (monitoring Phe1093 in cbEGF12)

cbEGF13 23 mM 6.5 [40] NMR

cbEGF13 27 mM 6.5 [39] As tandem cbEGF12cbEGF13; 2D NMR (monitoring Tyr1136 in cbEGF13)

cbEGF14 100 mM 6.5 [40] As tandem cbEGF13cbEGF14; NMR

cbEGF32 4 mM 7.4 [41] NMR (monitoring Tyr2149)

cbEGF32 9.2 mM 6.5 [42] As tandem cbEGF32cbEGF33; 2D NMR (monitoring Tyr2149 in cbEGF32)

cbEGF33 0.35 mM 6.5 [42] As tandem cbEGF32cbEGF33; 2D NMR (monitoring Phe2188 in cbEGF33)

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.

Figure 4


G293

G375

Extracellular pH Endosomal pH

LDL

LDL

(a) (b)

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 (


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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

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 pointsallowing 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 filtration studies haveindicated that the extracellular domain of LDL-R indeed

has an extended shape at neutral pH, but is significantlymore compact at acidic pH [18]. In addition, the frag-

ment R1R4 does not associate with the rest of theextracellular domain at neutral pH, but it does at acidic

pH [18], supporting the idea of pH-dependent confor-

mations. Further biochemical and biophysical studies are

clearly needed to verify such a mechanism of intramole-cular 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 ligand-binding 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

ligandat acidic pH (either upon endocytosis or artificiallyat 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 thetruncated ligand-binding domain and the full-length

receptor, Davis et al. [6] concluded that components in

the EGF-precursor homology domain were specificallyneeded for acid-triggered ligand release in the endo-

somes. These results undermine the idea that, at endo-

somal pH, ligand release is dominated by a decreasein Ca2 affinity, but they are compatible with an intra-molecular displacement mechanism involving the b-

propeller domain.

ConclusionsThe 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 infor-

mation will lead to the design of targeted studies using

biophysical and mutagenesis techniques to confirm ourthinking on LDL-R function.

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

of special interestof outstanding interest

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An up-to-date review detailing genetic and biochemical aspects of LDL-R, and its relevance to the disease FH.

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12. Kurniawan ND, Atkins AR, Bieri S, Brown CJ, Brereton IM,Kroon PA, Smith R: NMR structure of a concatemer of the firstand second ligand-binding modules of the human low-densitylipoprotein receptor. Protein Sci 2000, 9:1282-1293.

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Kurniawan ND, Aliabadizadeh K, Brereton IM, Kroon PA, Smith R:NMR structure and backbone dynamics of a concatemer ofepidermal growth factor homology modules of the humanlow-density lipoprotein receptor. J Mol Biol 2001, 311:341-356.

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Crystallographic study revealing the structure of a fragment containingthe b propeller and the EGF-like repeat C at neutral Ph.

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Rudenko G, Henry L, Henderson K, Ichtchenko K, Brown MS,

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Structural insights into LDL receptor functioning Rudenko and Deisenhofer 689


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