FEBS Letters 589 (2015) 3534–3540

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The closed conformation of the LDL receptor is destabilized by the lowCa++ concentration but favored by the high Mg++ concentration in theendosome

http://dx.doi.org/10.1016/j.febslet.2015.10.0140014-5793/� 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Author contributions: Juan Martínez-Oliván: Did most experiments, wrote themanuscript; Xabier Arias-Moreno: Did cloning; Ramón Hurtado-Guerrero: Con-tributed to the expression, discussions and writing; José Alberto Carrodeguas:Contributed to the expression, discussions and writing; Laura Miguel-Romero:Contributed to the expression; Alberto Marina: Contributed to the expression,discussions and writing; Pierpaolo Bruscolini: Contributed to data analysis,discussions, and writing; Javier Sancho: Conceived and directed the work, wrotethe manuscript.⇑ Corresponding author at: Biocomputation and Complex Systems Physics

Institute (BIFI)-Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza,Spain. Fax: +34 976762123.

E-mail address: jsancho@unizar.es (J. Sancho).

Juan Martínez-Oliván a,b,c, Xabier Arias-Moreno a,b, Ramón Hurtado-Guerrero a,c,d,José Alberto Carrodeguas a,b,c, Laura Miguel-Romero e, Alberto Marina e, Pierpaolo Bruscolini a,Javier Sancho a,b,c,⇑aBiocomputation and Complex Systems Physics Institute (BIFI)-Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, SpainbDepartamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Zaragoza, SpaincAragon Health Research Institute (IIS Aragón), Universidad de Zaragoza, Zaragoza, Spaind Fundación ARAID, Diputación General de Aragón, Spaine Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (IBV-CSIC), Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 July 2015Revised 6 October 2015Accepted 14 October 2015Available online 23 October 2015

Edited by Lukas Huber

Keywords:Low density lipoprotein receptorSurface plasmon resonanceBindingEndosome

The LDL receptor (LDLR) internalizes LDL and VLDL particles. In the endosomes, it adopts a closedconformation important for recycling, by interaction of two modules of the ligand binding domain(LR4–5) and a b-propeller motif. Here, we investigate by SPR the interactions between those twomodules and the b-propeller. Our results indicate that the two modules cooperate to bind the b-propeller. The binding is favored by low pH and by high [Ca++]. Our data show that Mg++, at high con-centration in the endosome, favors the formation of the closed conformation by replacing the struc-turing effect of Ca++ in LR5. We propose a sequential model of LDL release where formation of theclose conformation follows LDL release.� 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

The low density lipoproteins (LDL) receptor (LDLR) plays a keyrole in internalization of LDL and VLDL remnants in the liver andperipheral tissues [1,2]. The LDLR binds circulating LDL and VLDLparticles, and is internalized in endosomes where the cargolipoprotein is released for degradation in lyzosomes, while thereceptor is recycled to the cell surface. Defects in the LDLR function

result in elevated concentrations of circulating cholesterol and arethe major cause of familial hypercholesterolemia (FH) [3–5]. TheLDLR [6] is a transmembrane protein constituted by five structuraldomains (Fig. 1): an N-terminal ligand binding domain (LBD), anEGF-like domain, a small glycosylated domain, a transmembranedomain that anchors the receptor to the membrane and aC-terminal cytoplasmic domain that plays an important role inlocalization and endocytosis of the LDLR [7–10]. The ligand bindingdomain, essential for the binding of LDL and VLDL remnants[11,12], is formed by seven homologous repeats (LR1 to LR7), eachcontaining about 40 residues, including 3 disulfide bonds and acalcium coordinating site that help maintaining the LR’s nativestructure [13–16]. The LR repeats, that can fold autonomously[17–19], are connected by small 4–6 residue flexible linkers.Multiple studies point to the central LR domains, LR4 and LR5, con-nected by a larger 12-residue linker, as the most important ones forcellular internalization and endosomal release of lipoproteins[6,12,20–23]. The EGF-like domain consists of two EGF-like repeats(EGFA and EGFB), a six-bladed b-propeller motive and an addi-tional EGF-like module (EGFC) [24–26]. This domain participates

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Fig. 1. The LR4–5/b-propeller interface. (a) Scheme of the LDL receptor. LR corresponds to Ligand Repeat; EGF to Epidermal Growth Factor; Gly to Glycosylated Domain; T toTransmembrane Domain and Cyt to Cytoplasmatic Domain. The cartoon and surface representation of the LR4–5 (PDB:2LGP) and the EGFB-b-propeller-EGFC (PDB:1IJQ) havebeen drawn with Pymol. Residues in LR4–5 that make hydrophobic contacts with the b-propeller in the closed conformation of the LDLR are colored in green and residuesinvolved in salt bridges, in yellow. Residues in the EGFB-b-propeller-EGFC that make hydrophobic contacts with the LR4–5 in the closed conformation of the LDLR are coloredin blue and residues involved in salt bridges, in red. Residues that make both hydrophobic and electrostatic contacts are shown in brown. (b) Closed conformation of the LDLR(PDB:1N7D). The same color scale as in panel (a) was used. (c) Zoom of the LR4–5/b-propeller interface shown in panel (b).

J. Martínez-Oliván et al. / FEBS Letters 589 (2015) 3534–3540 3535

in lipoprotein release in the endosome and receptor recycling tothe membrane [27–32].

The LDLR adopts an extended conformation when it is anchoredto the cell membrane and is exposed to extracellular conditions,which are characterized by a neutral pH and high (1–2 mM) Ca++

concentrations. However, the X-ray structure of the extracellularregion of the LDLR at high Ca++ concentration but acidic pHrevealed a closed conformation (Fig. 1b and c) stabilized by electro-static and hydrophobic contacts established between the LR4 andLR5 domains of the LBD ant the b-propeller of the EGF-like domain[6]. It has been proposed that the binding of the b-propeller to theLR4 and LR5 domains could facilitate LDL and VLDL remnantrelease by displacing the lipoproteins attached to the LBD or bypreventing rebinding of the lipoproteins dissociated in the endo-some [6,33–37]. One major issue here is whether the contactsobserved in the crystal structure are maintained under physiolog-ical conditions on the endosomes, where calcium concentrationdrops to the low micromolar range while magnesium concentra-tion remains around 1.5 mM [38,39]. To clarify it, we studied bysurface plasmon resonance (SPR) the interaction of the isolatedLR4 and LR5 domains and of the LR4–5 tandem (comprising theLR4 and LR5 domains joint by their physiological linker) with theb-propeller in different pH, Ca++ and Mg++ concentrations charac-teristic of either the extracellular or endosomal conditions.

2. Materials and methods

2.1. Cloning, expression and purification of LR4, LR5 and LR4–5

The LR4, LR5 and LR4–5 peptides were cloned as previouslydescribed [22,23,40,41]. They were expressed in Escherichia colicells as recombinant proteins fused to glutathione-sulfhydryltransferase (GST). The intercalated thrombin cleavage site adds,after cleavage, two amino acids (GS) at the N-terminal of LR4–5and LR4 and introduces a single substitution D172G on LR5 [41].

Typically, competent E. coli BL21 cells were transformed,selected and grown in LB medium, expression of the recombinantgene induced with isopropyl 1-thio-b-D-galactopyranoside (IPTG)and the corresponding protein purified by glutathione-agaroseaffinity chromatography followed by thrombin (GE Healthcare)cleavage at 18 �C. Once cut from the GST, the LR domains wererefolded under conditions permitting disulfide exchange. A finalpurification step was performed by RP-HPLC on a preparativeC18 column (Watters) [22,23,40,41]. A single peak was obtainedfor each protein. Purity > 99% was confirmed by silver-stainedSDS–PAGE and the identity of the disulfide bonds was verified byMALDI-TOF MS [40,41]. LR concentrations were determined usingtheir aqueous theoretical extinction coefficients.

2.2. Cloning, expression and purification of the EGFB-b-propeller-EGFC

The cDNA fragment coding for the EGFB-b-propeller-EGFC (BbC)of the LDLR was cloned in the pEF1/V5-His B plasmid (Invitrogen).The sequence corresponding to the exportation signal of the LDLRwas added on the N-terminal and a 6-His tag was incorporated onthe C-terminal of the protein. Expression was performed tran-siently in HEK 293 GNT�/� cells. Cultures in Free Style 293 med-ium (Gibco) were transfected with plasmidic DNA containing theconstruct for the BbC using linear 25 kD PEI (Sigma) as transfectingreagent. After 96 hours expression, cell cultures were harvestedand centrifuged. Cleared medium was passed through a Ni++ affin-ity column HisTrap FF 16/10 (GE Healthcare). The eluate was con-centrated and dialyzed against 100 mM Tris-HCl, 150 mM NaCl,pH 8.5 and the protein was purified in an exclusion size column(Superdex 200, GE Healthcare). All purification steps were per-formed at 4 �C. A single peak was isolated after gel filtration elec-trophoresis and a single band was obtained after SDS–PAGEelectrophoresis or western blot analysis using an anti-His HRPantibody (GenScript). Trypsinization of the sample followed byMALDI-TOF MS analysis confirmed the identity of the BbC protein

3536 J. Martínez-Oliván et al. / FEBS Letters 589 (2015) 3534–3540

and 1H NMR analysis showed a good dispersion signal, consistentwith a well folded protein (not shown).

2.3. Surface plasmon resonance experiments

Immobilization of the BbC in a CM5 chip was performed byamine coupling using the Amine Coupling Kit (GE Healthcare).The protein was dissolved in 10 mM sodium acetate buffer, pH5.0, and 10 mM HEPES buffer, 150 mM NaCl, 3 mM EDTA, 0.005%v/v surfactant P20, pH 7.4, was used as running buffer. Immobiliza-tion level reached was 493.4 RU.

Surface Plasmon Resonance (SPR) experiments were performedin a Biacore T200 instrument (GE Healthcare) at 25 �C. Binding ofLR4, LR5 or LR4–5 to the BbC was analyzed by injecting the ana-lytes (LR4, LR5, LR4–5 or LR4 and LR5 together, which we nameLR4 + LR5) at a flow rate of 30 lL/min for 60 s. The dissociationwas followed for 250 s by passing running buffer through the chipsurface. Using a regeneration solution was not necessary. Experi-ments were performed at pH 7.0 in 10 mM HEPES buffer, contain-ing 150 mMNaCl (extracellular conditions) and at pH 5.5 in 10 mMSodium Acetate buffer containing 150 mM NaCl (endosomal condi-tions). Either buffer contained 0.25 mM EDTA. In some experi-ments either 1.5 mM CaCl2 or 1.5 mM MgCl2 was additionallyincluded. To avoid non-specific interactions between the analyteand the surface of the binding chip, 1 mg/ml Bovine Serum Albu-min (BSA) was added to the running and injection buffers. Typi-cally, duplicated experiments with up to 5 different analyteconcentrations were performed and data were corrected for bulkand instrumental artefacts with the BIAevaluation 2.0 software(GE Healthcare) by double referencing to buffer injections and acontrol sensor chip surface activated with EDC/NHS and blockedwith 1 M ethanolamine. The association and dissociation processeswere too fast to be adjusted properly by our kinetic analysis per-formed with either the BIAevaluation 2.0 software or with home-made scripts implemented in Mathematica (Wolfram). In the rangeof analyte concentrations tested, we were unable to reach satura-tion levels of the reaction and, therefore, the mathematical analysisof the data is somewhat limited [42]. We have used the percentageof the maximum signal response level expected (Rmax) reached asan indication of the binding under different conditions. Rmax corre-sponds to the signal response value expected when all the immo-bilized BbC molecules on the chip are attached to a molecule ofanalyte (either LR4, LR5 or LR4–5). Rmax was calculated as:

Fig. 2. Comparison between LR4–5, LR4, LR5 and LR4 + LR5 binding to the b-propeller. Peto immobilized b-propeller after 60 s of injection in a buffer containing 10 mM sodium aBSA with, where indicated, either 1.5 mM CaCl2 or 1.5 mM MgCl2. *LR4 + LR5 binding w

Rmax ¼ RL �MWA � nMWLwhere RL represents the amount of ligand immobilized, MWA andMWL are the molecular weights of analyte and ligand respectivelyand n represents the stoichiometric binding coefficient that wasassumed to be 1 for each LR. When injecting LR4 + LR5, one mole-cule of each LR can bind simultaneously one immobilized BbC. Thissimple analysis reveals important differences in the binding of thedifferent LR peptides to the b-propeller in the solution conditionstested.

3. Results and discussion

3.1. LR4–5 binds the b-propeller cooperatively

Surface Plasmon Resonance experiments performed by addingfrom 4 to 60 lM LR4–5 to a CM5 chip where the BbC constructwas immobilized showed interaction between the two molecules.When experiments were performed at pH 5.5 in presence of1.5 mM CaCl2, 74.2% of the BbC molecules on the chip wereattached to an LR4–5 molecule after 60 s of injection of 60 lMLR4–5 (Figs. 2 and 3, Table 1). In the same conditions, 22.5% ofthe Rmax value expected was observed when 60 lM LR4 is addedand only 3.0% when the experiment was performed with 60 lMLR5 (Figs. 2 and 3, Table 1).

The increased binding observed for the LR4–5 tandem constructcompared to that for the isolated LR4 and LR5 repeats is indicativeof a chelating effect. Binding of one of the LR modules to BbC bringsthe other LR module in close proximity, which seems to facilitatethe overall association. Furthermore, once LR4–5 is bound, itsrelease from BbC requires the dissociation of both LR4 and LR5,which slows down dissociation compared to that of the individualrepeats (see Fig. 3). The use of a chelating effect strategy toenhance the affinity of LR4 and LR5 to other biological partnerssuch as LDL and b-VLDL has been previously proposed [21,22].An alternative explanation for the greater binding observed forLR4–5 compared to that observed for either isolated LR4 or LR5would be that the binding of one LR module induces a conforma-tional change in the b-propeller that favors the binding of the otherLR. To evaluate this possibility, we performed experiments whereisolated LR4 and LR5 were added simultaneously to the BbC. When60 lM LR4 plus 60 lM LR5 were simultaneously added, 15.6% of

rcentage of Rmax reached in SPR experiments when performing 60 lM LR injectionscetate (pH 5.5) or HEPES (pH 7.0) buffer, 150 mM NaCl, 0.25 mM EDTA and 1 mg/mlas only tested at pH 5.5 in presence of 1.5 mM CaCl2.

Fig. 3. Binding of LR4, LR5 and the LR4–5 pair to the b-propeller. Traces of LR4–5, LR4, LR5 and LR4 + LR5 binding to immobilized b-propeller analyzed by SPR. 4 (black),8 (orange), 16 (light blue), 24 (purple), 30 (blue), 40 (green), 60 (red), 80 (navy) and 120 lM (dark green) analyte concentrations were recorded. In all cases, experiments wererecorded in a buffer containing 10 mM Sodium Acetate (pH 5.5) or HEPES (pH 7.0) buffer, 150 mM NaCl, 0.25 mM EDTA and 1 mg/ml BSA with, where indicated, either1.5 mM CaCl2 or 1.5 mM MgCl2.

Table 1LR/b-propeller binding. Percentage of Rmax reached in 60 lM LR injections to immobilized b-propeller after 60 s of injection.1

pH 5.5 pH 7.0

CaCl2 (1.5 mM) MgCl2 (1.5 mM) – CaCl2 (1.5 mM) –

LR4-5 74.2 ± 4 33.1 ± 1 17.1 ± 0.3 19.2 ± 3 0.7 ± 0.1LR4 22.5 ± 0.5 5.6 ± 0.4 3.5 ± 0.1 7.3 ± 0.1 –LR5 3.0 ± 0.2 5.4 ± 0.6 2.1 ± 0.4 1.4 ± 0.3 –

1 All solutions contain 0.25 mM EDTA.

J. Martínez-Oliván et al. / FEBS Letters 589 (2015) 3534–3540 3537

the Rmax signal value was reached after 60 s of interaction (Figs. 2and 3, Table 1). This signal, much lower than that achieved for theLR4–5 tandem (74.2%), is quite similar to the average of thoseobtained for the isolated LR4 and LR5 modules (12.8%) and indi-cates that there is no binding cooperativity if the modules arenot linked. Therefore, conformational changes in BbC after bindingof one LR module are not a likely cause of the enhanced bindingobserved for the LR4–5 tandem.

Interestingly, LR4 seems to bind to BbC much better than LR5(Figs. 2 and 3, Table 1). As previously shown, LR4 plays an impor-tant role in the binding of lipoproteins [20,21]. Its reduced Ca++

affinity and lower conformational stability compared to those ofLR5 [18,23,41,43] suggest that, when pH and Ca++ concentrationdecrease in the endosome, the dissociation of ApoE and ApoB fromLR4 probably precedes their dissociation from LR5 [18,22,41]. Ourdata now suggests, although do not prove, that after thelipoproteins are released from the receptor, binding of LR4 to theb-propeller may precede that of LR5, as previously suggested byZhao and coworkers [31].

3.2. High Ca++ and low pH enhance the binding of LR domains to theb-propeller

Studies by Rudenko et al. by gel filtration and dynamic lightscattering showed that, at pH 5, the LDLR displays a hydrodynamicradius corresponding to a monomer with a 95 kD apparent masswhile at pH 8, the hydrodynamic radius corresponds to a proteinof 160 kD [6]. Additionally, they showed that, after cleavage ofthe LR1-LR4 fragment, it remained attached to the rest of the mole-cule at pH 5.5 but not at pH 8. This experiments, and the X-raystructure solved at pH 5.3 [6], suggest that the closed conformationof the LDLR occurs only at acidic pH and explanations have beenoffered that relate such higher binding at a lower pH with changesin the protonation of specific residues [6,28,31,32,35,37,44]. Our

results indicate that binding of LR4–5 to the b-propeller at pH7.0 is significantly decreased compared to its binding at pH 5.5.In presence of 1.5 mM CaCl2, only 19.2% of the Rmax is reached after60 s of injection of a 60 lM LR4–5 sample which is more than 3times lower than the binding observed at pH 5.5 in the same con-ditions (Figs. 2 and 3, Table 1). Similarly, the LR4 binding level inpresence of 1.5 mM CaCl2 decreases 3 times (from 22.5% to 7.3%of Rmax) when going from pH 5.5 to pH 7.0 and LR5 binding isreduced by a factor of 2 (from 3.0% to 1.4%) (Fig. 2, Table 1). Thisresults correlate well with the biological observations shownpreviously and demonstrate in a direct way, using a simplifiedmolecular model, the effect exerted by pH changes on the LR4–5/b-propeller binding strength.

Unlike what has been discussed for the effect of pH, a relation-ship between calcium concentration and the adoption of a closedor open structure in the LDLR did not become evident when theextracellular structure of the receptor was solved. Studies in ourgroup showed that the conformational stability of apoLR5 is signif-icantly lower than that of the LR5/Ca++ form and we proposed that,in endosomal conditions, Ca++ release from LR5 and probably otherrepeats could lead to their unfolding and trigger the release of thelipoproteins [23]. Presumably, the unfolding of LR repeats may alsohinder its binding to the b-propeller domain. Recent works haverevealed that LR4, due to its low calcium affinity and low confor-mational stability, would be essentially present as unfoldedapoLR4 in endosomal conditions [18,41] and that the closed struc-ture of the LDL receptor is disrupted at low calcium concentrationsat pH 6.0 [29].

Our SPR experiments reveal that, in absence of calcium, bindingof LR4–5 to the BbC fragment is markedly reduced (Figs. 2 and 3,Table 1). At pH 5.5, with 0.25 mM EDTA, 60 lM of LR4–5 saturate17.1% of the immobilized ligands after 60 s, 4 times less than thebinding observed in presence of 1.5 mM CaCl2 under the sameconditions (Figs. 2 and 3, Table 1). Similarly, under the same

3538 J. Martínez-Oliván et al. / FEBS Letters 589 (2015) 3534–3540

conditions, LR5 binding is slightly reduced without calcium, from3.0% to 2.1% of Rmax (Fig. 2, Table 1), and LR4 binding is drasticallyreduced from 22.5% with calcium to 3.5% without the cation (Fig. 2,Table 1). Not surprisingly, depletion of calcium causes a greatereffect on the binding of LR4 than on that of LR5, consistent withthe larger conformational change reported for LR4 when it unfoldsand releases Ca++ at pH 5.5 [41].

As it appears, both high calcium concentrations and low pH pro-mote binding of LR domains to the b-propeller. At pH 7.0, with0.25 mM EDTA, only residual binding to the b-propeller is observedfor the LR4–5 pair, reaching 0.7% of Rmax after 60 s injection ofLR4–5 (Figs. 2 and 3, Table 1), and we could not detect bindingof the b-propeller to the isolated LR4 or LR5 modules (Fig. 2,Table 1).

3.3. Mg++ can partially replace the function of Ca++ in the endosome

Acidic pH contributes to lipoprotein release in the endosome byweakening the interaction between LR5, and presumably LR4, tothe binding regions of ApoE and ApoB in b-VLDLs and LDLs, respec-tively [22] and by improving the interactions between the LR4–5pair domain and the b-propeller, as we have discussed above. Incontrast, decrease of calcium concentration in the endosome playsa dual role. On one hand, it weakens the interactions betweenapolipoproteins and LR domains [22], which favors lipoprotein dis-sociation. On the other, it seems to lower the affinity of LR4–5 forBbC, which might interfere with subsequent formation of theclosed LDLR conformation.

We showed previously that magnesium may replace the cal-cium ion at the coordination site of LR5 although with somewhatreduced affinity [41,43]. In contrast, LR4 is not able to bind magne-sium at physiological concentrations [41]. Here we show that LR4–5 binding to the b-propeller at pH 5.5 in presence of 0.25 mM EDTA(i.e. absence of Ca++) is clearly increased upon addition of 1.5 mMMgCl2. However, such high Mg++ concentration is not as effectiveas the same Ca++ concentration. At 60 lM, after 60 s of injection,

Fig. 4. LDLR cycle. The LDLR binds LDL or VLDL particles in the extracellular space and theendosome because drops in [Ca++] and pH promote the unfolding of the LR4–5 pair doapolipoproteins present in LDLs or VLDLs. Afterwards, the LDLR adopts a closed conformfavored by the low pH and the presence of Mg++, that can replace the Ca++ previously releMg++ and may be partly unfolded. Then, the LDLR is recycled to the cellular membrane whand LR5. In the figure, LR1 to LR7 are represented as purple circles and the LR4 and LR5(marked as C) are represented as blue squares and the b-propeller (marked as b) asrepresented as a green diamond, a yellow rectangle and an orange diamond respectively.little red squares.

74.2%, 33.1% and 17.1% of Rmax are reached when 1.5 mM CaCl2,1.5 mM MgCl2 or only 0.25 mM EDTA, respectively, are present(Figs. 2 and 3, Table 1). We have analyzed the individual bindingresponse of the LR4 and LR5 domains to the presence of Mg++ ions.Very little difference between magnesium and EDTA-containingexperiments is observed for LR4. At 60 lM LR4, in 60 s, 22.5%,5.6% and 3.5% of the surface is saturated when 1.5 mM CaCl2,1.5 mM MgCl2 or 0.25 mM EDTA respectively are present (Fig. 2,Table 1). In contrast, similar binding of LR5 is observed with eithermagnesium or calcium. At endosomal pH, 3.0% and 5.4% of Rmax isreached when 1.5 mM CaCl2 or MgCl2 is added and 1.4% when theexperiments are performed with only 0.25 mM EDTA (Fig. 2,Table 1).

According to these results, the coordination of Mg++ to LR5 mayallow the LR4–5 tandem to recover part of its ability to bind theb-propeller. This is physiologically relevant because, unlike forCa++, the magnesium concentration in the endosome hardlychanges and it remains similar to the extracellular concentration[38,39].

3.4. Role of the EGF homologous domain in lipoprotein release andLDLR recycling

Calcium-dependent and acid-dependent mechanisms cooperateto release lipoproteins in the endosome (Fig. 4) [29]. The impor-tance of the EGF homologous domain for the acid-dependentrelease was first demonstrated by experiments with mutatedreceptors bearing a deletion of the BbC region [27]. Afterwards,the X-ray structure of the closed conformation of the LDLR[6,28,33,34,37] provided a plausible explanation of the role of theEGF homologous domain, which has been confirmed by recentworks [29,31,32,44]. Acidic pH also destabilizes the interactionsbetween LR5, and probably other binding domains such as theLR4, and the ApoE and ApoB in the LDL and VLDL particles(Fig. 4) [22]. Our data here suggest that the EGF homologousdomain does not participate in the calcium-dependent mechanism

LDLR/Lipoprotein complex is internalized. The LDLR releases the lipoproteins in themain and the concomitant destabilization of interactions between LR4–5 and theation where important contacts between LR4–5 and the b-propeller are established,ased form LR5. In this close conformation LR4 is not expected to carry either Ca++ orere high pH favors the extended conformation and high [Ca++] replenishes both LR4domains are marked as 4 and 5 respectively; EGFA, EGFB (marked as B) and EGFCa blue circle. The glycosylated, transmembrane and cytoplasmatic domains areThe LDLs or VLDLs are represented as a red circle and their degradation products as

J. Martínez-Oliván et al. / FEBS Letters 589 (2015) 3534–3540 3539

of release because the drop of calcium concentration in theendosome is detrimental for the formation of the LR4–5/b-propeller complex. Likewise, it has been found that LDLR lackingthe BbC region supports calcium-dependent release of LDL and b-VLDL with very similar rates to the wild type receptors [29].

The LR4–5 tandem uses essentially the same surface residuesfor binding lipoproteins when the LDLR exhibits its open confor-mation and for binding the b-propeller domain in the LDLR closedconformation [6,22]. Considering the large size of LDLs and b-VLDLs it is difficult to envision how the b-propeller residues thatwill eventually appear bound to LR4–5 in the closed conformationof the LDLR could actively displace the bound lipoproteins, as thiswould require a cis attack on the severely occluded LR4–5 bindingsite. When endosomes are formed, the drop in calcium concentra-tion, that decreases to 7 lM in 5 min, is faster than the drop in pH,that falls to 6.0 in 10 min [38], so it seems reasonable to assumethat the calcium-dependent mechanism acts first. We thus proposethat release of lipoproteins from the LDLR takes place after the keyLR binding modules, which include LR4 and LR5, lose calcium andbecome unfolded. This is a plausible mechanism sustained bykinetic and thermodynamic studies of the LR4 and LR5. The afore-mentioned decrease in calcium concentration lowers the fractionof folded LR5-Ca++ from 99.6% to 40% and that of folded LR4-Ca++

from 62% to 0.2% [41] (the lower affinity of LR4 for calcium, andhence its more extensive unfolding compared to LR5, has beenexplained in structural terms [41]). It is likely that other LR repeatsexperience calcium dissociation and unfolding in the endosomebut the relevance of such processes to the mechanism of LDLrelease is uncertain [37,45]. On the other hand the kinetics of cal-cium dissociation from either LR5 or LR4 are relatively fast koffbeing 3.70 and 10.5 s�1, respectively [41], which means that thefractions of unfolded apoLR5 and apoLR4 are rapidly adjusted tothe changing endosomal calcium concentration. Once the lipopro-tein particle is released, the b-propeller binds to the vacant LR4–5to form the closed structure of the LDLR. This binding will befavored by Mg++ coordination to apoLR5 (but not to apoLR4)(Fig. 4). The affinity of magnesium for apoLR5 is of 643 lM and,at the endosomal magnesium concentration, the fraction of mag-nesium bound apoLR5 is of around 31% [41]. The binding of mag-nesium to apoLR5 is very fast (kon = 0.0047 lM�1 s�1) and takesplace in seconds [41]. The generally lower affinity of magnesiumfor apoLR4 and apoLR5 compared to that of calcium [41] is relatedto its lower ionic radius not allowing an efficient coordination bythe repeats, as previously shown in MD simulations [41,43]. Oncethe closed conformation of the LDLR is formed, it impairs rebindingof lipoproteins to the receptor, enhancing the overall lipoproteinrelease rate and preventing the recycling to the membrane ofligand-containing receptors (Fig. 4) [31,32]. Additionally, the adop-tion of the closed structure has been reported to be important forLDLR recycling because it blocks the binding to the LDLR of PCSK9,a protease which promotes LDLR trafficking to the proteasome fordegradation [46–48].

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The closed conformation of the LDL receptor is destabilized by the low Ca++ concentration but favored by the high Mg++ concentration in the endosome1 Introduction2 Materials and methods2.1 Cloning, expression and purification of LR4, LR5 and LR4–52.2 Cloning, expression and purification of the EGFB-β-propeller-EGFC2.3 Surface plasmon resonance experiments

3 Results and discussion3.1 LR4–5 binds the β-propeller cooperatively3.2 High Ca++ and low pH enhance the binding of LR domains to the β-propeller3.3 Mg++ can partially replace the function of Ca++ in the endosome3.4 Role of the EGF homologous domain in lipoprotein release and LDLR recycling

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