LDL Receptorlipoprotein Recognition: Endosomal Weakening of Apob And

LDL receptor/lipoprotein recognition: endosomal weakening of ApoB and ApoE binding to the convex face of the LR5 repeat Juan Martınez-Olivan 1,2, Xabier Arias-Moreno1,2, Adrian Velazquez-Campoy1,2,3, Oscar Millet4 and Javier Sancho1,2

1 Biocomputation and Complex Systems Physics Institute (BIFI). BIFI-Instituto de Quımica Fısica Rocasolano (Consejo Superior de Investigaciones Cientıﬁcas) Joint Unit, Universidad de Zaragoza, Spain 2 Departamento de Bioquımica y Biologıa Molecular y Celular, Universidad de Zaragoza, Spain 3 Fundacion Agencia Aragonesa para la Investigacion y Desarrollo, Diputacion General de Aragon, Spain 4 Structural Biology Unit, CIC bioGUNE, Derio, Spain

Keywords The molecular mechanism of lipoprotein binding by the low-density lipopro- ApoB; ApoE; low-density lipoprotein (LDL) tein (LDL) receptor (LDLR) is poorly understood, one reason being that receptor; NMR; SPR structures of lipoprotein–receptor complexes are not available. LDLR uses calcium-binding repeats (LRs) to interact with apolipoprotein B and apoli- Correspondence J. Sancho, Departamento de Bioquımica y poprotein E (ApoB and ApoE). We have used NMR and SPR to character- Biologı´a Molecular y Celular, Facultad de ize the complexes formed by LR5 and three peptides encompassing the Ciencias, Universidad de Zaragoza, 50009 putative binding regions of ApoB (site A and site B) and ApoE. The three Zaragoza, Spain peptides bind at the hydrophilic convex face of LR5, forming complexes that Fax: +34 976762123 are weakened at low [Ca2+] and low pH. Thus, endosomal conditions favour Tel: +34 976761286 dissociation of LDLR/lipoprotein complexes regardless of whether active E-mail: [email protected] displacement of bound lipoproteins by the b-propeller in LDLR takes place. b b (Received 5 September 2013, revised 2 The multiple ApoE copies in very low density lipoproteins ( -VLDLs), and January 2014, accepted 13 January 2014) the presence of two competent binding sites (A and B) in LDLs, suggest that LDLR chelates lipoproteins and enhances complex afﬁnity by using more doi:10.1111/febs.12721 than one LR.

Introduction The receptor of low-density lipoproteins (LDLR) is a seven homologous LR repeats, each comprising multi-domain transmembrane protein of 839 residues approximately 40 residues and including three disulfide [1,2] that belongs to a family of membrane receptors bonds and a structural calcium ion [7,8]. The fifth LR that include the VLDL receptor, apoER2, MEGF7, repeat (LR5) appears to play a major role in lipopro- LRP, LRP1B and the Megalin receptor [3], all of tein recognition [4,5]. which are formed by various combinations of a few Interaction of low-density lipoprotein (LDL) parti- common domains. The primary ligands of LDLR are cles with LDLR is mediated by a single copy of apoli- the cholesterol-rich LDL and b-VLDL lipoproteins poprotein B (ApoB), which, with 4536 residues, is one [4,5], and its chief role is maintaining cholesterol of the largest proteins known [9,10]. Although the size homeostasis. Many reported mutations in the gene and complexity of ApoB have prevented a detailed encoding LDLR lead to familial hypercholesterola- structural characterization, its binding region has been emia, a disease that affects 1 in 500 persons worldwide identified as being located around residues 3356–3368 [6]. The ligand-binding domain of LDLR contains (termed site B), which are exposed on the surface of

Abbreviations ApoB, apolipoprotein B; ApoE, apolipoprotein E; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; LR, calcium-binding repeat of LDLR; RAP, receptor-associated protein; SPR, surface plasmon resonance.

1534 FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS J. Martınez-Olivan et al. Binding of apolipoproteins to the LDLR

LDL particles [11]. Additional binding sites have been of ApoE3 bind LR5 with similar affinity (KD values of proposed, such as site A (residues 3143–3155), 12, 23 and 56 lM for site A, site B and ApoE3 pep- although their relevance has been questioned [12–14]. tides). The binding takes place at a common site in In addition to one copy of ApoB, b-VLDLs include LR5 that includes some of the residues involved in cal- several copies of the smaller apolipoprotein E (ApoE) cium binding together with several conserved residues (299 residues) [15,16]. The C-terminal domain of ApoE of previously unknown function. anchors the protein to the lipid particle, while the N- terminal domain is a four-helix bundle [17] that binds Results and Discussion LDLR through it fourth helix [18–20] and additional residues [21]. LR5 binds apolipoprotein peptides at Total de-lipidation of ApoE and ApoB leads to pro- extracellular pH in a Ca2+-dependent manner tein aggregation [17,22], which complicates structural studies of their binding to LDLR. So far, it has been Performing surface plasmon resonance (SPR) experi- shown that positively charged peptides with sequences ments at 25 °C allowed us to determine the association found in ApoE bind to LDLR [23,24] or LRP recep- and dissociation rate constants (kon and koff) and the tors [25], a docking model of the ApoE(135–151)/LR5 equilibrium dissociation constant (KD) for binding of complex has been proposed [26], and weak interactions LR5 to the three apolipoprotein peptides. Although all between LR3, LR4 and LR5 and a linear fragment of three peptides bind to LR5 and dissociate from it fast ApoE have been described [27,28]. However, detailed which complicates determination of the rates, accurate structural information for the complexes formed by values have been derived for each peptide complex by ApoB and ApoE with LDLR is lacking. global fitting of various traces obtained at various pep- LR5 is one of the most important repeats within the tide concentrations (see Experimental procedures). Pre- ligand-binding domain of LDLR [5,29,30]. We have vious to this analysis, we first discarded the possibility described the oxidative folding pathway of wild-type that the association observed between the apolipopro- LR5 repeat and mutant LR5 repeats found in familial tein mimetic peptides and LR5 is non-specifically dri- hypercholesterolaemia [31], and have proposed that ven by electrostatic interactions by analysing a the structural instability of LR5 under endosomal con- randomized peptide with the same amino acid composi- ditions favours LDL release in the endosome [32,33]. tion as site A of ApoB. The SPR response of the ran- Here, we determine the affinity of LR5 for peptides domized peptide (see binding traces at several pH comprising putative binding sequences of ApoB and values in Fig. S1) is almost negligible compared to that ApoE. At extracellular pH, peptides representing of the ApoB site A peptide or the two other mimetic site A and site B from ApoB and the binding region peptides (Figs 1–4).

Fig. 1. ApoB site B peptide binding to LR5 under extracellular pH conditions (NaCl/Pi buffer, pH 7.4) in the presence of either

250 lM CaCl2 (A, B) or 1 mM EDTA (C, D). (A, C) SPR association and dissociation curves of the ApoB site B peptide at 25 lM (dark green), 35 lM (blue), 50 lM CD (brown), 70 lM (pink), 100 lM (green) and 200 lM (black). (B, D) The initial association step, with the experimental traces indicated by black triangles and their global ﬁtting to Eqns (1–3) (see Experimental procedures) indicated by red lines. All experimental points in the initial 5 s are shown, but only one in ten experimental points is shown for the rest of the trace.

FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS 1535 Binding of apolipoproteins to the LDLR J. Martınez-Olivan et al.

Fig. 2. ApoB site A peptide binding to LR5

under extracellular pH conditions (NaCl/Pi buffer, pH 7.4) in the presence of either

250 lM CaCl2 (A, B) or 1 mM EDTA (C, D). (A) SPR association and dissociation curves of the ApoB site A peptide at 15 lM (dark green), 25 lM (blue), 35 lM (brown), 50 lM (pink) and 100 lM (green). (C) SPR association and dissociation CD curves of the ApoB site A peptide at 13 lM (dark green), 27 lM (blue), 54 lM (brown), 108 lM (pink) and 217 lM (green). (B, D) The initial association step, with the experimental traces indicated by black triangles, and their global ﬁtting to Eqns (1–3) (see Experimental procedures) indicated by red lines. All experimental points in the initial 5 s are shown, but only one in ten experimental points is shown for the rest of the trace.

Fig. 3. ApoE3 peptide binding to LR5

under extracellular pH conditions (NaCl/Pi buffer, pH 7.4) in the presence of either

250 lM CaCl2 (A, B) or 1 mM EDTA (C, D). (A) SPR association and dissociation curves of ApoE3 peptide at 10 lM (dark green), 15 lM (blue), 25 lM (brown), 40 lM (pink), 70 lM (green) and 100 lM (black). (C) SPR association and dissociation CD curves of ApoE3 peptide at 25 lM (dark green), 35 lM (blue), 50 lM (brown) and 150 lM (green). (B, D) The initial association step, with the experimental traces indicated by black triangles, and their global ﬁtting to Eqns (1–3) (see Experimental procedures) indicated by red lines. All experimental points in the initial 5 s are shown, but only one in ten experimental points is shown for the rest of the trace.

The importance of the structural calcium ion in the KD values in the micromolar range (Table 2). The bind- interaction of LDLR with lipoproteins was highlighted ing/dissociation kinetics of the site B peptide at various by the observation that the calcium chelator EDTA dis- peptide concentrations are shown in Fig. 1, and those of rupts LDL and b-VLDL binding to LDLR [34]. We site A and ApoE3 peptides are shown in Figs 2 and 3. tested whether the site A, site B or ApoE3 mimicking Calcium removal does not abolish binding of the pep- peptides (Table 1) bind to LR5 under otherwise extra- tides to LR5, but significantly reduces the affinity of cellular conditions lacking calcium (NaCl/Pi, pH 7.4, each of these complexes (Table 2). Site A, site B and 1mM EDTA). In the presence of calcium, the three apo- ApoE3 peptides differ in terms of binding kinetics and lipoprotein mimetic peptides bind LR5 with observed with regard to the influence exerted by Ca2+ on the

1536 FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS J. Martınez-Olivan et al. Binding of apolipoproteins to the LDLR

Fig. 4. ApoB site B peptide binding to LR5 at pH 7.0, 6.5, 6.0 or 5.5 in the presence of 250 lM CaCl2 and 150 mM NaCl. (A) SPR association and dissociation curves of the ApoB site B peptide at 12.5 lM (red), 17.5 lM (orange), 25 lM (dark green), 35 lM (blue), 50 lM (brown), 70 lM (pink), 100 lM (green) and 150 lM (cyan). (B) The initial association step, with the experimental traces indicated by black triangles, and their global ﬁtting to Eqns (1–3) (see Experimental procedures) indicated by red lines. All experimental points in the initial 5 s are shown, but only one in ten experimental points is shown for the rest of the trace.

Table 1. Sequences of apolipoprotein mimetic peptides. For ApoE3, a Q156A substitution was introduced within the 141–158 ApoE sequence to increase the peptide helix content.

Apolipoprotein Peptide Sequence sequence

ApoE3 WGLRKLRKRLLRDADDLAKR WG-(141-158 ApoE3) ApoB site B WGRLTRKRGLKLATA WG-(3356-3368 ApoB) ApoB site A WGVKAQYKKNKHRHS WG-(3143-3155 ApoB) Control WGSQVTQELRALMDETMKEL WG-(54-71 ApoE3) Randomized ApoB site A KWRNSHGKVKYQHAK –

2+ Table 2. Binding and dissociation rate constants, and dissociation ApoB peptide binds to LR5-Ca faster (kon = 0.031 1 1 equilibrium constants of complexes formed by three apolipoprotein lM s ) than it binds to ApoLR5 (kon = 0.010 1 1 mimetic peptides and LR5 under extracellular conditions (NaCl/Pi lM s ). In contrast, there appears to be no calcium l buffer, CaCl2 250 M, pH 7.4). dependence regarding the binding kinetics of site B Presence ApoB peptide. On the other hand, the complexes 2+ 1 1 1 Peptide of Ca kon (lM s ) koff (s ) KD (lM) formed by either site A or site B peptides with LR5- 2+ k Ca show similar off values that are increased (faster ApoE3 Yes 0.129 0.060 2.97 0.01 23 8 2+ ApoE3 No 0.011 0.001 2.15 0.09 190 30 dissociation) in the absence of Ca , especially for the ApoB site B Yes 0.011 0.001 0.59 0.01 55 3 site A complex, while the ApoE3/LR5 complex dissoci- ApoB site B No 0.013 0.003 1.58 0.05 126 7 ates more rapidly than those of the site A or site B pep- ApoB site A Yes 0.031 0.009 0.40 0.06 13 3 tides and in a calcium-independent manner. The ApoB site A No 0.010 0.001 2.90 0.1 300 20 combined effect of the binding and dissociation rate constants determines that calcium increases the afﬁnity of the complexes by factors of 2.5 (site B peptide), 8 afﬁnity of their complexes with LR5. The ApoE3 pep- (ApoE3 peptide) and 25 (site A peptide), as shown in 2+ 1 1 tide binds fastest to LR5-Ca (kon = 0.129 lM s ), Fig. 5A. That the peptides still bind LR5 in the absence and its rate of binding decreases tenfold when calcium is of Ca2+ may be surprising in view of the reported ran- 1 1 not present (kon = 0.011 lM s ). Similarly, the site A dom coil conformation of ApoLR5 [35], and may be

FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS 1537 Binding of apolipoproteins to the LDLR J. Martınez-Olivan et al.

A The hydrophilic convex face of LR5 is the lipoprotein-binding site Elucidation of the LDLR sequence [37] led to the initial proposal that the complex between the receptor and ApoE was stabilized by electrostatic interactions established by the negatively charged repeats of LDLR and a positively charged sequence found in ApoE. This proposal was generalized to the LDLR/LDL complex when multiple positively charged segments were found in the ApoB sequence [38,39]. However, the crystal structure of LR5 [8] showed that the conserved acidic residues of the repeat (D196, D200, D206 and E207) are essentially buried and are involved in direct binding of a structural B calcium ion. Two faces were observed in the LR5 surface: a hydrophilic convex face enclosing the calcium- binding pocket, and a hydrophobic concave face that was proposed to constitute the lipoprotein-binding site [8]. However, indirect support for involvement of the hydrophilic convex face of LR5 in lipoprotein binding was later provided by the X-ray structure of the extracellular domain of LDLR at endosomal pH [29], by the structure of the complex between RAP and LR3/LR4, in which the convex faces of several repeats appear to be involved in protein binding to RAP [40], and by model- ling work on binding of LR5 to an ApoE fragment [26]. More recently, there have been contradictory reports supporting a major role in lipoprotein binding for either Fig. 5. Influence of Ca2+ concentration and pH on the stability of the hydrophobic concave face [41,42] or for the hydro- mimetic peptide/LR5 complexes. (A) ApoE3, ApoB site B and ApoB philic convex face [27,43,44]. Further complicating this K site A equilibrium dissociation constants ( D values) in NaCl/Pi scenario, a possible role of the b-propeller domain of l buffer, pH 7.4, in the presence of either 250 M CaCl2 (light grey) LDLR in LDL binding has also been proposed [43,45]. or 1 mM EDTA (dark grey). (B) ApoB site B equilibrium dissociation To determine experimentally which face of LR5 constants (KD values) in 10 mM PIPES buffer in the presence of binds lipoproteins, we performed a solution NMR 250 lM CaCl2 and 150 mM NaCl at pH 7.0, 6.5, 6.0 and 5.5. chemical shift perturbation analysis using 15N-labelled LR5. Initially, a perturbation analysis was performed related to a stabilizing effect of the peptides on the at 25.0 °C, but only small changes in chemical shift structure of the repeat. It should be noted that the were observed (data not shown). According to AGA- site A, site B and ApoE3 peptides correspond to protein DIR predictions [46] and circular dichroism spectra segments that are thought to adopt helical conforma- (data not shown), the helical content of the synthesized tions in the apoprotein context. Because the helicities of peptides approximately doubles as the temperature free peptides are typically much lower than those exhib- decreases from 25.0 to 5.0 °C. We thus increased the ited within the corresponding proteins [36], it is expected peptide helical content by reducing the temperature, that the affinities for ApoB and ApoE exhibited by LR5 and performed chemical shift perturbation experiments are greater than those reported for the peptides in at 5.0 °C. Binding of either site A, site B or ApoE3 Table 2. This SPR analysis clearly demonstrates the peptides to LR5-Ca2+ produces significant changes in sequence-specific, non-electrostatically driven interac- the chemical shift of several LR5 residues (Fig. 6). In tion between apolipoprotein peptides and LR5. Further contrast, the presence of a non-binding control peptide analysis (e.g. by a split-ubiquitin assay or cellular (Table 1) does not perturb LR5-Ca2+ chemical shifts uptake of a fluorescent peptide in competition with an (Fig. 6). Inspection of the LR5 X-ray structure [8] indi- inhibitory ligand, such as the receptor-associated pro- cates that the perturbed residues are clustered in the tein (RAP) is required to confirm the interaction and three-dimensional structure. Thus, they may be identi- mechanisms in the cell. fied as the binding site common to ApoB and ApoE.

1538 FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS J. Martınez-Olivan et al. Binding of apolipoproteins to the LDLR

Fig. 6. (A) 1H–15N heteronuclear correlation map for the LR5 domain (black) and the same protein in the presence of the ApoB site B peptide (red). Numbers correspond to the assigned peaks in the sequence, and straight lines have been added to clarify chemical shift changes. (B) Chemical shift perturbation by the three peptides considered in the present study (ApoE, ApoB site B and ApoB site A qpeptides)ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi and a control peptide. The chemical shift perturbation shown on the y axis was calculated using the expression Ddð Þ2 þ c c Ddð Þ2 c and c are the gyromagnetic constants for nitrogen and proton, respectively. The grey bars highlight H N H N , where N H the concurrent interacting sites for the three peptides.

AB C

DE F

Fig. 7. Residues from LR5 implicated in binding of ApoB site B (A–C), ApoB site A (D–F) and ApoE3 (G–I) under extracellular conditions (20 mM phosphate buffer, pH GH I 7.5, with 8% D2O and 3 mM CaCl2). The residues with chemical shifts strongly modiﬁed upon peptide addition are shown in white. The remaining residues in LR5 are shown in green. The structural Ca2+ ion is shown in yellow.

The LR5 residues implicated in ApoB site B, ApoB tides, while the region around E180, F181, H182 and site A and ApoE3 binding are highlighted in Fig. 7. C183, also appears to be important in the ApoE3 and As judged from the observed chemical shift perturba- site A complexes. All these residues are located at the tions, W193, G197, G198, D200 and D206 appear to hydrophilic convex face of LR5, and we propose that be similarly important for binding of the three pep- they constitute a general lipoprotein-binding site in

FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS 1539 Binding of apolipoproteins to the LDLR J. Martınez-Olivan et al.

LR5. This lipoprotein-binding site essentially coincides residues (P175, E180, H182, S185, E187, H190, W193 with the sites used by LR5 and by the homologous and G197) that are conserved within vertebrate LR5 LR3/LR4 repeats to bind the LDLR b-propeller repeats but not among the seven human repeats. The domain and RAP, respectively [29,40]. This involve- reason for the conservation of P175 is not obvious. ment of the convex face of LR repeats in recognizing S185 and E187 play a structural role, being involved different but related proteins emphasizes the challenge in the hydrogen bond network mentioned above [8,48], of understanding how the affinity for its partners is H190 has been implicated in the mechanism of LDL modulated along the LDLR cellular route [47]. Other release [29,49], and E180, H182, W193 and G197 are repeats of the receptor, in addition to LR5, are likely precisely located at the lipoprotein-binding site to use a similar binding site to interact with lipopro- (Fig. 2). Residue W193 appears to be particularly teins, as suggested by binding of LR repeats to a ubiq- important, playing the dual role of contributing to cal- uitinated peptide derived from ApoE [27]. cium coordination and to apolipoprotein binding. Trp residues are well suited to form stabilizing cation/p interactions [50,51] with the positively charged residues Conserved residues of LR5 repeats from present in the three lipoprotein peptides analysed here. vertebrates are crucial for lipoprotein binding The key role played by calcium-coordinating trypto- Conserved residues of homologous proteins are typi- phan residues of LDLR repeats in the interaction cally involved in protein stabilization or protein func- established by the receptor with other proteins has tion. All the conserved residues of the seven LR been highlighted [47], and its importance in the bind- repeats in human LDLR (Fig. 8) play distinct roles in ing of lipoproteins has also been demonstrated by maintaining the stability of the modules: the six cyste- mutational studies [42,43]. At present, it is unclear ines form three disulfide bonds, F181 and I189 form a whether the interaction of W193 with LDL or other small hydrophobic core, D196, D200, D206 and E207 proteins influences the coordination of the calcium- coordinate the structural calcium ion, and D203 is binding site. It is also possible that the contribution of important in maintaining a hydrogen bond network other residues, such as G198, to calcium coordination connecting the two lobes of the repeats [8,48]. Given may be influenced by the binding of proteins to LR5. the clear structural role of these residues and the fact that not all the repeats are equally important for lipo- LR5 binding to ApoB site B peptide is weaker at protein binding, it is not surprising that some of the endosomal pH: implications for the mechanism lipoprotein-binding residues identified in LR5 are not of LDL release conserved among the repeats. They are nevertheless conserved among LR5 repeats from different species. We tested binding of the three lipoprotein peptides to Alignment of LR5 sequences for a number of LR5 under endosomal mimicking conditions (10 mM vertebrates from human to fish (Fig. 8) reveals several MES, 150 mM NaCl, pH 5.5) by SPR. However, two

Fig. 8. Sequence alignment between LR5 from various species (upper panel) and between the seven repeats (LR1–LR7) of the ligand-binding domain in the Homo sapiens LDLR (lower panel) performed using ClustalW (http:// embnet.vital-it.ch/software/ClustalW.html). Conserved residues are coloured using the ClustalW code, i.e. green (polar), red (apolar) and blue (acidic), while non- conserved residues are shown in black.

1540 FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS J. Martınez-Olivan et al. Binding of apolipoproteins to the LDLR of the three apolipoprotein peptides (ApoB site A and uted to lower peptide helicity, it must be concluded ApoE) could not be analysed at this pH due to non- that endosomal pH weakens the binding of native specific interactions with the surface of the Biacore LR5-Ca2+ to the ApoB site B peptide. The molecular chips that could not be alleviated by increasing the reason for this cannot be related to weakening of cat- ionic strength or changing the buffer, Fortunately, for ion/p interactions between W193 and basic residues of the apoB site B peptide, we were able to analyse fine the peptide because Trp/Lys and Trp/Arg interactions binding/dissociation traces at several pH values: 7.0, are similarly strong at pH 7.5 and 5.0, while Trp/His 6.5, 6.0 and 5.5, which evaluation of the effect of the interactions are even stronger at pH 5.0 [50,51] than at endosomal decrease in pH on the affinity of its com- pH 7.5. Thus, the most likely reasons for weakening of plex with LR5. The experimental traces and their fits the complex at low pH are either weakening of charge/ to Eqns (1–3) are shown in Fig. 4, and the fitted val- charge interactions due to protonation of LR5 carb- ues of kon, koff and KD at the various pH values are oxylates, or protonation of H190 and/or H182 if they shown in Table 3. The affinity of the complex is mark- are buried in the complex without forming a suitable edly reduced as the pH is decreased. At pH 7.4, under stabilizing interaction. very similar but not identical solution conditions, the The weakening of the ApoB peptide binding to LR5

KD was 55 lM (Table 2). The data in Table 3 indicate at low pH has a bearing on the mechanism of LDL that, at pH 7.0, the KD is 142 lM and progressively release in the endosome. The currently accepted model increases to 854 lM at pH 5.5 (see also Fig. 5B). The for LDL release states that the LDL particles that lower affinity at lower pH is mainly due to faster dis- bind to LR repeats at the cell surface are displaced by sociation of the complex at low pH, with the increase the b-propeller of LDLR, which, under endosomal in koff being noticeable between pH 6.5 and 6.0. The conditions, displays a higher affinity for the repeats fact that the affinity of the complex is reduced as a than LDL [29,49]. It may also be argued that a role consequence of faster dissociation rather than of for the b-propeller in LDL release is demonstrated by slower formation is relevant because the direct effect mutational experiments indicating that, when this of reducing the pH is to favour dissociation of previ- domain is removed, internalized LDLR is not recycled ously formed LDL/LDLR complexes. Complementary to the cell surface [52]. However, the mechanism of evidence for weakening of the ApoB site B/LR5 com- LDLR recycling is not fully understood, and a lack of plex at low pH has been obtained by NMR. We first recycling need not be incompatible with LDL/LDLR tried to use chemical shift perturbation analysis to esti- dissociation. More recent work implying specific mate the dissociation constants, but no plateau was involvement of the propeller in LDL release at low pH found in the experimental range tested. Nevertheless, [42,43] was performed in the presence of Ca2+. we were able to determine the slopes of the variation Our observation that only a small fraction of the of the chemical shift with the equivalents of titrant LR5 population is in the native Ca2+-complexed form (7.5 and 13 ppbeq1 at pH 5.0 and 7.5, respectively, under endosomal conditions is suggestive of an alter- data not shown). These numbers indicate that binding native mechanism of LDL release based on reversible of ApoB site B to natively folded LR5-Ca2+ is binding/folding events [33]. We proposed that LDL reduced at low pH (5.0) compared with extracellular release may be simply triggered by LR5 destabilization pH conditions. at the low pH and low calcium concentrations that are The helical content of the three peptides is the same found in the endosome [33]. An admitted weak point at pH 5.0 and 7.5 according to AGADIR [46]. As the in our proposal was that the potential influence of weaker binding observed at low pH cannot be attrib- lipoprotein binding on the stability of LR5 was not examined, and therefore the possibility that LR5 is far more stable in complex with LDL than in isolation Table 3. Binding and dissociation rate constants, and dissociation cannot be excluded. However, our present analysis of equilibrium constants of complexes formed by the ApoB site B LR5 binding to lipoprotein mimicking peptides sug- peptide and LR5 at various pH values (10 mM PIPES buffer, gests the possibility that LDL/LDLR complexes may l 2+ 150 mM NaCl, 250 M CaCl2). no longer be stable at endosomal pH and Ca con-

1 1 1 centration. Endosomal conditions appear to result in Peptide pH kon (lM s ) koff (s ) KD (lM) dissociation of the LDL/LDLR complex by reducing ApoB site B 7.0 0.016 0.001 2.28 0.04 142 20 the afﬁnity of LR5 for ApoB (this work) and unfold- ApoB site B 6.5 0.012 0.001 2.17 0.03 183 20 ing the LR5 repeat [31], which displaces further the ApoB site B 6.0 0.010 0.002 4.47 0.2 430 130 equilibrium towards dissociation. It is nevertheless ApoB site B 5.5 0.008 0.002 6.96 0.4 854 400 unclear at present, and remains to be tested, whether

FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS 1541 Binding of apolipoproteins to the LDLR J. Martınez-Olivan et al. the complexes formed by LDLR and LDL, which are presumably stronger than those formed between LR5 and the peptides analysed here, are similarly disrupted by endosomal pH conditions.

Proposal for multivalent binding of LDLR to LDL and b-VLDL It is generally accepted that the LDLR-binding region of ApoE is the fourth helix, although additional residues have been reported as important for binding [21]. To some extent, the observed binding of the ApoE3 peptide to LR5 was expected. In contrast, the binding of the site A peptide to LR5 was not antici- pated. When the sequence of ApoB was published [9,38,39,53], several basic regions were singled out as potential binding sites for the LDLR [9,11,38,39,53,54], and two of them (site A, residues 3147–3157; site B, residues 3359–3369) were identified as the main candidates. Phylogenetic studies [14] sug- Fig. 9. Schematic representation of proposed multivalent binding gested that site B was the primary region involved in of LDL and b-VLDL to the LDLR. In b-VLDL binding, LR5 interaction with the LDLR, but also attributed an recognizes one molecule of ApoE (A), and then additional ApoE molecules in the particle bind to another repeat (B). In LDL binding, important role to site A in LDL binding to LDLR. an interaction is first established between LR5 and either ApoB Experiments with truncated LDLs that contain either site B or ApoB site A (probably site B) (C), and an additional repeat both site A and site B [55] or neither [56] confirmed binds the additional free site of ApoB (probably site A) (D). that the sequence encompassing both site A and site B is required for LDLR binding, while subsequent studies with LDL lacking the site B sequence [13] indicated this site by another LR in the receptor. However, that site A alone is not sufficient for LDL binding. given the scarcity of kinetic data, the existence of alter- Mutational experiments [12] also indicated that site B native kinetic binding mechanisms cannot be excluded, is indeed necessary for receptor binding. and additional investigation is required. However, it has been shown here (Table 2) that, The observed affinity of the ApoE3/LR5-Ca2+ com- under extracellular conditions, a peptide encompassing plex may similarly be too low to justify efficient bind- the sequence of site A binds LR5 with higher affinity ing. We thus propose that b-VLDL particles use more than a site B peptide. Possible explanations for the than one copy of ApoE in order to bind simulta- observed inability of LDLs lacking site B to effectively neously to LR5 and additional repeats of LDLR, con- bind to LDLR may be that site A is not accessible in sistent with reported observations of formation of low- the mature lipoprotein, or that additional LDL/LDLR affinity complexes between LR4 and LR5 and a ubiq- interactions are required to ensure effective binding. uitinated ApoE peptide [27,28]. Although the affinities of sites A and B for LR5 within In summary, our kinetic and structural analysis indi- the context of the entire ApoB protein are probably cates that the binding of three apolipoprotein mimetic higher than the micromolar affinities exhibited in isola- peptides (ApoB site A, ApoB site B and ApoE) to tion, it is uncertain whether the nanomolar affinities LR5 is fast, takes place at the convex hydrophilic face characteristic of high-affinity complexes are achieved. of LR5, and it is modulated by pH and calcium con- In this respect, an efficient way to increase the affinity centration. We propose that the binding of LDL and of the LDL/LDLR complex would be to make use of b-VLDL by LDLR is multivalent. the chelating effect. It seems likely that site A and site B bind LDLR by associating with LR5 plus one Experimental procedures additional repeat (Fig. 9), greatly increasing the overall affinity of the LDL/LDLR complex. A sequential Cloning and purification of LR5 binding model may be envisaged in which site B binds first to LDLR. Then, re-organization in the LDL leads The fragment corresponding to the fifth repeat of the LDLR to a greater exposure of site A, allowing recognition of gene was amplified by PCR and cloned into pGEX-4T-3

1542 FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS J. Martınez-Olivan et al. Binding of apolipoproteins to the LDLR

(Amersham Biosciences, Little Chalfont, UK), and recombi- 100–350 lM concentration in 20 mM phosphate buffer (pH nant LR5 was expressed and purified as described previ- 7.5) with 8% D2O and 3 mM CaCl2.At5C, two-dimen- ously [33]. Pure, reduced, unfolded LR5 was re-folded by sional 1H–15N-HSQC spectra were recorded in the absence exhaustive dialysis under conditions permitting disulfide and the presence of 2.5 equivalents of ApoE3, site A or exchange, and further purified by reverse-phase HPLC in a site B peptides. Spectral assignment at 278 K was based on Symmetry C18 column (Waters, Milford, MA, USA). Silver the published assignment at 5 C [35], corroborated by staining after SDS/PAGE showed the LR5 was 99% pure. analysis of a 3D-HSQC-NOESY spectrum of LR5-Ca2+. MALDI-TOF MS using a Bruker Ultraflex mass spectrome- All chemical shifts were estimated from the 1H-15N-HSQC ter confirmed the purity of the repeat and the uniqueness of spectra by interpolation of the peak centre using NMRPipe the disulfide pattern [31]. A theoretical extinction coefficient [58] and scripts written in-house. 1 1 (e280) of 6050 M cm was used in determination of LR5 concentrations [57]. Surface plasmon resonance experiments

Isotope labelling for NMR studies SPR experiments were performed in a Biacore T200 instrument (GE Healthcare, Fairﬁeld, CT, USA) at 25 °C. The plasmid containing the fragment of the gene corre- Lyophilized LR5 was immobilized on a CM5 chip by sponding to LR5 was transformed into competent Escheri- amine coupling according to the manufacturer’s instruc- chia coli BL21 cells. Colonies were grown in ampicillin- tions using HBS/EP (10mM Hepes, 150mM NaCl, 3mM containing LB agar plates for 30 h. Selected colonies were EDTA, 0.005% Tween-20) as running buffer. One ﬂow cell grown in LB medium (10 mL) for 5 h. The harvested cells was activated with EDC/NHS (N-(3-dimethylaminopropyl)- were resuspended in minimal medium in which the only N’-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide), 15 1 nitrogen source was NH4Cl (1 gL ), and cultured in blocked with 1 M ethanolamine, and used as a control sur-

2 L Erlenmeyer flasks (typically containing 1 L of culture face. Experiments were performed in NaCl/Pi buffer, pH ° with ampicillin) at 37 C. Induction was performed after 7.4, with either 250 lM CaCl2 or 1 mM EDTA. The pep- approximately 10 h by adding 1 mM isopropyl thio-b-D- tides (ApoE3, site A and site B peptides) were injected at a galactoside. Purification of labelled LR5 was performed as flow rate of 30 lLmin1 for 40 s, and dissociation steps described for the unlabelled protein. were followed for 10 min. Typically, four to six peptide concentrations were used for each measurement. Similar experiments were performed with ApoB site B peptide and Design of peptides encompassing apolipoprotein the randomized ApoB site A peptide in 10 mM PIPES buf- sequences fer, 150 mM NaCl, 250 lM CaCl2 at pH 7.0, 6.5, 6.0 and The five peptides used in this work were synthesized by 5.5 with 0.1 mgmL 1 BSA (Sigma-Aldrich, St. Louis, MO, Pompeu Fabra University (Spain) and ChinaPeptides USA) to avoid non-specific interactions. Data analysis was (Shanghai, China), and are summarized in Table 1. The performed using software developed in our laboratory and site A and site B peptides mimic the ApoB sequences tradi- implemented in MLAB. The SPR signal was considered to tionally known as site A (residues 3143–3155) and site B consist of the binding/dissociation signal plus the bulk- (residues 3356–3368). The ApoE3 peptide mimics ApoE effect signals affecting each of the two steps. The associa- (141–158), and contains a Q156A substitution to increase tion and dissociation events were analysed separately, con- the a-helix propensity. The control peptide mimics residues sidering their corresponding bulk effects as very fast 54–71 of ApoE, a segment of the protein that is located in exponential signals. Thus, the dissociation part of each the first helix of the bundle and is not expected to bind to trace was fitted to: LR5. The randomized ApoB site A peptide has the same kobst kbulkt composition as the ApoB site A peptide but with a random S ¼ Aoff e þ Abulk e þ Send ð1Þ order of the original amino acid residues. An N-terminal

Trp residue followed by a Gly was added to each peptide to where S is the signal detected by the instrument, Aoff is 1 1 allow peptide quantification [57] (e280 = 5500 M cm for the signal change attributed to LR5/peptide dissociation, e = 1 1 ApoE3, site B and control peptides; 280 6990 M cm kobs is the apparent dissociation rate constant, Abulk is for the site A peptide and the randomized ApoB site A pep- the amplitude of the bulk effect, kbulk is the rate con- tide). The five peptides are acetylated and amidated, respec- stant of the bulk effect, and Send refers to the SPR sig- tively, at their N- and C-termini. nal at the end of the dissociation process. Because the experiments were performed using a continuous flow of running buffer, the dissociation observed is an irreversible NMR experiments process where kobs = koff. Therefore, the zero-order disso- All experiments were performed using a Bruker Avance ciation rate constant (koff) may be directly obtained from 600 MHz spectrometer with LR5 15N-labelled samples of the fitting.

FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS 1543 Binding of apolipoproteins to the LDLR J. Martınez-Olivan et al.

Similarly, the association part of the curves was fitted to: binding of low density lipoprotein receptor to two different proteins. J Biol Chem 264, 21682–21688. kobst kbulkt 6 Voight BF, Peloso GM, Orho-Melander M, Frikke- S ¼ Aon e þ Abulk e þ Send ð2Þ Schmidt R, Barbalic M, Jensen MK, Hindy G, Holm where Aon is the signal change attributed to LR5/peptide H, Ding EL, Johnson T et al. (2012) Plasma HDL binding, Abulk and kbulk are the amplitude and rate constant cholesterol and risk of myocardial infarction: a of the bulk effect, respectively, Send refers to the SPR signal mendelian randomisation study. Lancet 380, 572–580. at the end of the dissociation process, and kobs is the appar- 7 Daly NL, Scanlon MJ, Djordjevic JT, Kroon PA & ent association rate constant, which is related to the micro- Smith R (1995) Three-dimensional structure of a scopic kon and koff by: cysteine-rich repeat from the low-density lipoprotein receptor. Proc Natl Acad Sci USA 92, 6334–6338. k ¼ k ½Pþk ð3Þ obs on off 8 Fass D, Blacklow S, Kim PS & Berger JM (1997) where [P] represents the concentration of free peptide. Molecular basis of familial hypercholesterolaemia from Nature – For each of the three peptides investigated, the traces structure of LDL receptor module. 388, 691 693. obtained at various peptide concentrations were globally 9 Chen SH, Yang CY, Chen PF, Setzer D, Tanimura M, fitted to Eqns (1) or (2) plus (3) in order to derive common Li WH, Gotto AM Jr & Chan L (1986) The complete k , k and k values for the various traces. In the case cDNA and amino acid sequence of human on off bulk – J Biol Chem – of the randomized ApoB site A peptide, very minor bind- apolipoprotein B 100. 261, 12918 12921. € € ing was observed and fitting was not performed. 10 Hevonoja T, Pentikainen MO, Hyvonen MT, Kovanen PT & Ala-Korpela M (2000) Structure of low density lipoprotein (LDL) particles: basis for understanding Acknowledgements molecular changes in modified LDL. Biochim Biophys Acta 1488, 189–210. We acknowledge financial support from grants 11 Weisgraber KH & Rall SC Jr (1987) Human BFU2010-16297, BFU2010-19451, CTQ2009-10353 apolipoprotein B–100 heparin-binding sites. J Biol from Spain and Grupo Protein Targets B89 from the Chem – 262, 11097 11103. Diputacion General de Aragon (Spain). X.A.-M. was 12 Boren J, Lee I, Zhu W, Arnold K, Taylor S & supported by a Basque Government fellowship. We Innerarity TL (1998) Identification of the low density thank S. Bronsoms (Proteomics facility, Universitat lipoprotein receptor-binding site in apolipoprotein B100 Autonoma de Barcelona) for analysis of MALDI-TOF and the modulation of its binding activity by the data, and S.C. Blacklow (Department of pathology, carboxyl terminus in familial defective apo-B100. J Clin Harvard University) for the NMR assignments of the Invest 101, 1084–1093. LR5 module. 13 Chen Z, Saffitz JE, Latour MA & Schonfeld G (1999) Truncated apo B–70.5-containing lipoproteins bind to J Clin Invest References megalin but not the LDL receptor. 103, 1419–1430. 1 Anderson RG, Goldstein JL & Brown MS (1976) 14 Law A & Scott J (1990) A cross-species comparison of Localization of low density lipoprotein receptors on the apolipoprotein B domain that binds to the LDL plasma membrane of normal human fibroblasts and receptor. J Lipid Res 31, 1109–1120. their absence in cells from a familial 15 Rall SC Jr, Weisgraber KH & Mahley RW (1982) hypercholesterolemia homozygote. Proc Natl Acad Sci Human apolipoprotein E. The complete amino acid USA 73, 2434–2438. sequence. J Biol Chem 257, 4171–4178. 2 Yamamoto T, Davis CG, Brown MS, Schneider WJ, 16 Breslow JL, McPherson J, Nussbaum AL, Williams Casey ML, Goldstein JL & Russell DW (1984) The HW, Lofquist-Kahl F, Karathanasis SK & Zannis VI human LDL receptor: a cysteine-rich protein with (1982) Identification and DNA sequence of a human multiple Alu sequences in its mRNA. Cell 39,27–38. apolipoprotein E cDNA clone. J Biol Chem 257, 3 Beffert U, Stolt PC & Herz J (2004) Functions of 14639–14641. lipoprotein receptors in neurons. J Lipid Res 45, 17 Wilson C, Wardell MR, Weisgraber KH, Mahley RW 403–409. & Agard DA (1991) Three-dimensional structure of the 4 Esser V, Limbird LE, Brown MS, Goldstein JL & LDL receptor-binding domain of human Russell DW (1988) Mutational analysis of the ligand apolipoprotein E. Science 252, 1817–1822. binding domain of the low density lipoprotein receptor. 18 Innerarity TL, Friedlander EJ, Rall SC Jr, Weisgraber J Biol Chem 263, 13282–13290. KH & Mahley RW (1983) The receptor-binding domain 5 Russell DW, Brown MS & Goldstein LJ (1989) of human apolipoprotein E. Binding of apolipoprotein E Different combinations of cysteine-rich repeats mediate fragments. J Biol Chem 258, 12341–12347.

1544 FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS J. Martınez-Olivan et al. Binding of apolipoproteins to the LDLR

19 Lalazar A, Weisgraber KH, Rall SC Jr, Giladi H, module 5 of the low density lipoprotein receptor. J Biol Innerarity TL, Levanon AZ, Boyles JK, Amit B, Chem 283, 13627–13637. Gorecki M, Mahley RW et al. (1988) Site-specific 32 Arias-Moreno X, Cuesta-Lopez S, Millet O, Sancho J mutagenesis of human apolipoprotein E. Receptor & Velazquez-Campoy A (2010) Thermodynamics of binding activity of variants with single amino acid protein–cation interaction: Ca+2 and Mg+2 binding to substitutions. J Biol Chem 263, 3542–3545. the fifth binding module of the LDL receptor. Proteins 20 Weisgraber KH, Innerarity TL, Harder KJ, Mahley 78, 950–961. RW, Milne RW, Marcel YL & Sparrow JT (1983) The 33 Arias-Moreno X, Velazquez-Campoy A, Rodrıguez JC, receptor-binding domain of human Pocovı M & Sancho J (2008) Mechanism of low density apolipoprotein E. Monoclonal antibody inhibition of lipoprotein (LDL) release in the endosome: implications binding. J Biol Chem 258, 12348–12354. of the stability and Ca2+ affinity of the fifth binding 21 Morrow JA, Arnold KS, Dong J, Balestra ME, module of the LDL receptor. J Biol Chem 283, 22670– Innerarity TL & Weisgraber KH (2000) Effect of 22679. arginine 172 on the binding of apolipoprotein E to the 34 Schneider WJ, Basu SK, McPhaul MJ, Goldstein JL & low density lipoprotein receptor. J Biol Chem 275, Brown MS (1979) Solubilization of the low density 2576–2580. lipoprotein receptor. Proc Natl Acad Sci USA 76, 5577– 22 Walsh MT & Atkinson D (1983) Solubilization of low- 5581. density lipoprotein with sodium deoxycholate and 35 North CL & Blacklow SC (1999) Structural recombination of apoprotein B with independence of ligand-binding modules five and six of dimyristoylphosphatidylcholine. Biochemistry 22, 3170– the LDL receptor. Biochemistry 38, 3926–3935. 3178. 36 Sancho J & Fersht AR (1992) Dissection of an 23 Dyer CA, Cistola DP, Parry GC & Curtiss LK (1995) enzyme by protein engineering. The N and Structural features of synthetic peptides of C–terminal fragments of barnase form a native-like apolipoprotein E that bind the LDL receptor. J Lipid complex with restored enzymic activity. J Mol Biol Res 36,80–88. 224, 741–747. 24 Dyer CA & Curtiss LK (1991) A synthetic peptide 37 Sudhof€ TC, Goldstein JL, Brown MS & Russell DW mimic of plasma apolipoprotein E that binds the LDL (1985) The LDL receptor gene: a mosaic of exons receptor. J Biol Chem 266, 22803–22806. shared with different proteins. Science 228, 815–822. 25 Croy JE, Brandon T & Komives EA (2004) Two 38 Knott TJ, Pease RJ, Powell LM, Wallis SC, Rall SC Jr, apolipoprotein E mimetic peptides, ApoE(130–149) and Innerarity TL, Blackhart B, Taylor WH, Marcel Y, ApoE(141–155)2, bind to LRP1. Biochemistry 43, 7328– Milne R et al. (1986) Complete protein sequence and 7335. identification of structural domains of human 26 Prevost M & Raussens V (2004) Apolipoprotein E–low apolipoprotein B. Nature 323, 734–738. density lipoprotein receptor binding: study of protein– 39 Yang CY, Chen SH, Gianturco SH, Bradley WA, protein interaction in rationally selected docked Sparrow JT, Tanimura M, Li WH, Sparrow DA, complexes. Proteins 55, 874–884. DeLoof H, Rosseneu M et al. (1986) Sequence, 27 Guttman M, Prieto JH, Croy JE & Komives EA (2010) structure, receptor-binding domains and internal Decoding of lipoprotein–receptor interactions: repeats of human apolipoprotein B–100. Nature 323, properties of ligand binding modules governing 738–742. interactions with apolipoprotein E. Biochemistry 49, 40 Fisher C, Beglova N & Blacklow SC (2006) Structure 1207–1216. of an LDLR–RAP complex reveals a general mode for 28 Guttman M & Komives EA (2011) The structure, ligand recognition by lipoprotein receptors. Mol Cell dynamics, and binding of the LA45 module pair of the 22, 277–283. low-density lipoprotein receptor suggest an important role 41 Zhao Z & Michaely P (2011) Role of an intramolecular for LA4 in ligand release. Biochemistry 50, 11001–11008. contact on lipoprotein uptake by the LDL receptor. 29 Rudenko G, Henry L, Henderson K, Ichtchenko K, Biochim Biophys Acta 1811, 397–408. Brown MS, Goldstein JL & Deisenhofer J (2002) 42 Zhao Z & Michaely P (2008) The epidermal growth Structure of the LDL receptor extracellular domain at factor homology domain of the LDL receptor drives endosomal pH. Science 298, 2353–2358. lipoprotein release through an allosteric mechanism 30 Jeon H & Blacklow SC (2005) Structure and involving H190, H562, and H586. J Biol Chem 283, physiologic function of the low-density lipoprotein 26528–26537. receptor. Annu Rev Biochem 74, 535–562. 43 Huang S, Henry L, Ho YK, Pownall HJ & Rudenko G 31 Arias-Moreno X, Arolas JL, Aviles FX, Sancho J & (2010) Mechanism of LDL binding and release probed Ventura S (2008) Scrambled isomers as key by structure-based mutagenesis of the LDL receptor. intermediates in the oxidative folding of ligand binding J Lipid Res 51, 297–308.

FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS 1545 Binding of apolipoproteins to the LDLR J. Martınez-Olivan et al.

44 Guttman M, Prieto JH, Handel TM, Domaille PJ & 53 Cladaras C, Hadzopoulou-Cladaras M, Nolte RT, Komives EA (2010) Structure of the minimal interface Atkinson D & Zannis VI (1986) The complete sequence between ApoE and LRP. J Mol Biol 398, 306–319. and structural analysis of human apolipoprotein B–100: 45 Ren G, Rudenko G, Ludtke SJ, Deisenhofer J, Chiu W relationship between apoB–100 and apoB–48 forms. & Pownall HJ (2010) Model of human low-density EMBO J 5, 3495–3507. lipoprotein and bound receptor based on cryoEM. Proc 54 Marcel YL, Innerarity TL, Spilman C, Mahley RW, Natl Acad Sci USA 107, 1059–1064. Protter AA & Milne RW (1987) Mapping of human 46 Munoz V & Serrano L (1995) Elucidating the folding apolipoprotein B antigenic determinants. problem of helical peptides using empirical parameters. Arteriosclerosis 7, 166–175. II. Helix macrodipole effects and rational modification 55 Krul ES, Parhofer KG, Barrett PH, Wagner RD & of the helical content of natural peptides. J Mol Biol Schonfeld G (1992) ApoB–75, a truncation of 245, 275–296. apolipoprotein B associated with familial 47 Blacklow SC (2007) Versatility in ligand recognition by hypobetalipoproteinemia: genetic and kinetic studies. LDL receptor family proteins: advances and frontiers. J Lipid Res 33, 1037–1050. Curr Opin Struct Biol 17, 419–426. 56 Welty FK, Seman L & Yen FT (1995) Purification of 48 Cuesta-Lopez S, Falo F & Sancho J (2007) the apolipoprotein B–67-containing low density Computational diagnosis of protein conformational lipoprotein particle and its affinity for the low density diseases: short molecular dynamics simulations reveal a lipoprotein receptor. J Lipid Res 36, 2622–2629. fast unfolding of r–LDL mutants that cause familial 57 Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, hypercholesterolemia. Proteins 66,87–95. Williams KL, Appel RD & Hochstrasser DF (1999) 49 Beglova N, Jeon H, Fisher C & Blacklow SC (2004) Protein identification and analysis tools in the ExPASy Cooperation between fixed and low pH-inducible server. Methods Mol Biol 112, 531–552. interfaces controls lipoprotein release by the LDL 58 Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J

receptor. Mol Cell 16, 281–292. & Bax A (1995) NMRPipe: a multidimensional spectral 50 Loewenthal R, Sancho J & Fersht AR (1992) processing system based on UNIX pipes. J Biomol Histidine–aromatic interactions in barnase. Elevation of NMR 6, 277–293.

histidine pKa and contribution to protein stability. J Mol Biol 224, 759–770. 51 Fernandez-Recio J, Vazquez A, Civera C, Sevilla P & Supporting information Sancho J (1997) The tryptophan/histidine interaction in Additional supporting information may be found in a–helices. J Mol Biol 267, 184–197. the online version of this article at the publisher’s web € 52 Davis CG, Goldstein JL, Sudhof TC, Anderson RG, site: Russell DW & Brown MS (1987) Acid-dependent Fig. S1. Binding of the randomized ApoB site A pep- ligand dissociation and recycling of LDL receptor tide to LR5 in PIPES buffer at pH 7.0, 6.5, 6.0 or 5.5 mediated by growth factor homology region. Nature in the presence of 250 lM CaCl2 and 150 mM NaCl. 326, 760–765.

1546 FEBS Journal 281 (2014) 1534–1546 ª 2014 FEBS