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J. Biol. Chem., Vol. 275, Issue 28, 21017-21024, July 14, 2000

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Identification of the Minimal Functional Unit in the Low Density Lipoprotein Receptor-related Protein for Binding the Receptor-associated Protein (RAP)

A CONSERVED ACIDIC RESIDUE IN THE COMPLEMENT-TYPE REPEATS IS IMPORTANT FOR RECOGNITION OF RAP^*

Olav Michael Andersen, Lisa Lystbæk Christensen, Peter Astrup Christensen, Esben S. Sørensen^§, Christian Jacobsen^¶, Søren K. Moestrup^¶, Michael Etzerodt, and Hans Christian Thøgersen

From the  Laboratory of Gene Expression and ^§ Protein Chemistry Laboratory, Department of Molecular and Structural Biology, and the ^¶ Department of Medical Biochemistry, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark

Received for publication, January 24, 2000, and in revised form, March 22, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor family, mediates the internalization of a diverse set of ligands. The ligand binding sites are located in different regions of clusters consisting of ~40 residues, cysteine-rich complement-type repeats (CRs). The 39-40-kDa receptor-associated protein, a folding chaperone/escort protein required for efficient transport of functional LRP to the cell surface, is an antagonist of all identified ligands. To analyze the multisite inhibition by RAP in ligand binding of LRP, we have used an Escherichia coli expression system to produce fragments of the entire second ligand binding cluster of LRP (CR3-10). By ligand affinity chromatography and surface plasmon resonance analysis, we show that RAP binds to all two-repeat modules except CR910. CR10 differs from other repeats in cluster II by not containing a surface-exposed conserved acidic residue between Cys^IV and Cys^V. By site-directed mutagenesis and ligand competition analysis, we provide evidence for a crucial importance of this conserved residue for RAP binding. We provide experimental evidence showing that two adjacent complement-type repeats, both containing a conserved acidic residue, represent a minimal unit required for efficient binding to RAP.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The proteins of the low density lipoprotein receptor (LDLR)¹ family (reviewed in Ref. 1), are a group of related mosaic transmembrane receptors of similar structure and binding a diverse range of protein ligands in their ectodomains. Ligands bound to LDLR-like receptors are internalized by classical endocytosis (2). In humans, the group of known LDLR-like receptors includes the canonical LDLR (3), LRP (4, 5), the very low density lipoprotein receptor (VLDLR) (6), the apoE receptor2 (apoER2) (7), megalin/gp330 (8), and two recently discovered members, LRP6 (9) and LRP7 (10, 11).

The normal processing of LRP and megalin requires the presence of RAP (12), a 39-40-kDa protein (13) that appears to consist of three homologous domains (14-17) of which domain 1 has been shown to consist of a three-helix bundle (18). RAP interacts with all LDLR-like receptors and is a universal antagonist for all receptor/ligand interactions. RAP domains 1 and 3 (RAPd3) are both receptor-binding (15, 19), but only domain 3 is sufficient to mimic the chaperone-like functions of RAP in cells (20, 21). RAP domain 2 is a substrate for cAMP-dependent protein kinase (22) but has only a very low affinity for LRP and megalin compared with RAP domains 1 and 3 (23).

The ectodomain of LDLR members contains clustered complement-type repeats and epidermal growth factor precursor homology domains, consisting of multiple copies of cysteine-rich epidermal growth factor-like repeats and regions with a 6-fold YWTD consensus sequence, the latter suggested to form a compact -propeller domain (24). CRs consist of ~40 amino acids of which six are cysteines forming three disulfide bridges identified in all known repeats connecting Cys^I-Cys^III, Cys^II-Cys^V, and Cys^IV-Cys^VI (25-27).² The three-dimensional structure has been solved for three LDLR modules (27-29) and one LRP module (30), revealing an octahedral calcium cage that is formed by four conserved acidic residues plus two nearby carbonyl oxygens (27, 31).

A multitude of ligands are binding to these complement-type repeats, some exhibiting cross-competition such as, for example, tissue-type plasminogen activator and transformed ₂-macroglobulin (₂M*), while others do not. This suggests a variety of individual binding sites. So far, no reports have succeeded in identifying single residues as important for the ligand specificity of any LDLR-like receptor. Recently, Rong et al. (32) were able to substitute the fourth ligand binding repeat (LB4) of LDLR into the avian receptor for subgroup A Rous sarcoma virus (the Tva receptor), instead of the single naturally occurring complement-type repeat. They succeeded with only a few mutations converting LB4 residues to residues originally present in the Tva receptor repeat to obtain a chimeric receptor that functioned indistinguishably from the native Tva receptor. This engineered module has three mutations: Ala-19^LB4  Leu^Tva, Asp-23^LB4  His^Tva, and (Pro-34^LB4-Gln-35^LB4/Arg-36^LB4  Gly^Tva. Solving the crystal structure of LB5 of LDLR revealed that the residue corresponding to Asp-23^LB4 is located on the surface of the molecule with its backbone carbonyl group in a rigid position coordinating a calcium ion (27). The solution structure of CR8 from LRP also showed an aspartic acid residue located at the surface at this position (30). This is interesting, since ligand binding has previously been suggested to be dependent on charged residues, but the conserved acidic SDE-cluster, formerly speculated to bind positive charged ligands, is buried in the interior of the module and is in fact coordinating Ca²⁺.

The present study was undertaken to define and characterize the minimal functional unit in LRP binding RAP. Our molecular dissection of the second ligand binding cluster of CR modules of LRP and expression in Escherichia coli now delineate a minimal two-repeat RAP-binding unit and demonstrate the importance of a conserved acidic residue in each RAP-binding CR for the LRP high affinity binding of RAP.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of Expression Plasmids and Site-directed Mutagenesis-- A plasmid containing a complete LRP cDNA insert (kindly provided by Dr. J. Herz, University of Texas Southwestern Medical Center, Dallas, Texas) served as template in polymerase chain reactions (PCRs), using the following sets of primers to generate ubiquitin (U)-fused expression constructs encoding the four single-repeat derivatives CR3, CR4, CR5, and CR6 and the seven two-repeat derivatives CR34, CR45, CR56, CR67, CR78, CR89, and CR910 corresponding to CR pairs 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, and 9-10.

For U-CR3 (LRP residues Ala-844 to His-893), the primer pairs 5'-GGC GGA TCC ATC GAG GGT AGG GCG AAC CCA TCC TAC G-3' and 5'-GCC AAG CTT AGT GCT GAT GGC AGA GG-3'; for U-CR4 (LRP residues Gln-892 to Arg-934), the primer pairs 5'-GGC GGA TCC ATC GAG GGT AGG CAG CAC ACC TGC CCC-3' and 5'-GCC AAG CTT AGC GGG CTG AAC AAG TG-3'; for U-CR5 (LRP residues Ser-932 to Pro-974), the primer pairs 5'-GGC GGA TCC ATC GAG GGT AGG TCA GCC CGC ACC TGC-3' and 5'-GCC AAG CTT AGG GAT AGG CAC ACG AAG-3'; and for U-CR6 (LRP residues Tyr-973 to His-1013), the primer pairs 5'-GGC GGA TCC ATC GAG GGT AGG TAT CCC ACC TGC TTC-3' and 5'-GCC AAG CTT AGT GGC TGC AGC CGG-3' were used.

For U-CR34 (LRP residues Ala-844 to Arg-934), the primer pairs 5'-GGC GGA TCC ATC GAG GGT AGG GCG AAC CCA TCC TAC G-3' and 5'-GCC AAG CTT AGC GGG CTG AAC AAG TG-3'; for U-CR45 (LRP residues Gln-892 to Pro-974), the primer pairs 5'-GGC CGA TCC ATC GAG GGT AGG CAG CAC ACC TGC CCC-3' and 5'-GCC AAG CTT AGG GAT AGG CAC ACG AAG-3'; for U-CR56 (LRP residues Ser-932 to His-1013), the primer pairs 5'-GGC GGA TCC ATC GAG GGT AGG TCA GCC CGC ACC TGC-3' and 5'-GCC AAG CTT AGT GGC TGC AGC CGG-3'; for U-CR67 (LRP residues Tyr-973 to Arg-1057), the primer pairs 5'-GGC GGA TCC ATC GAG GGT AGG TAT CCC ACC TGC TTC-3' and 5'-GCC AAG CTT ACC TCG TGG CCT GGT TG-3'; for U-CR78 (LRP residues Ser-1012 to His-1102), the primer pairs 5'-GGC GGA TCC ATC GAG GGT AGG AGC CAC TCC TGT TCT AG-3' and 5'-GCC AAG CTT AGT GGG TCA CTC CCT C-3'; for U-CR89 (LRP residues Thr-1056 to Leu-1143), the primer pairs 5'-GGC GGA TCC ATC GAG GGT AGG ACG AGG CCC CCT GG-3' and 5'-GCC AAG CTT ACA GGG ACT CGC AGT TC-3', and for U-CR910 (LRP residues Gly-1099 to Gln-1184), the primer pairs 5'-GGC GGA TCC ATC GAG GGT AGG GGA GTG ACC CAC GTC-3' and 5'-GCC AAG CTT ACT GGT CGC AGA GCT C-3' were used.

Each set of primers was designed to introduce BamHI and HindIII restriction sites, a sequence encoding a factor X_a cleavage site and a stop codon at appropriate sites in the amplified products. PCR amplification products were digested with BamHI and HindIII and cloned into BamHI-HindIII-cut E. coli T7 expression vector pT7H6Ubi (15). Fusion proteins encoded by these vector constructs, pT7H6UbiFXCRxy, contain a hexahistidine affinity tag followed by residues 2-76 of human ubiquitin and a factor X_a recognition sequence preceding the receptor fragment, CRxy.

Mutations were performed using the Quickchange kit (Stratagene, La Jolla, CA) and pT7H6UbiFXCR34, pT7H6UbiFXCR45, or pT7H6UbiFXCR56 where appropriate as template for the mutagenesis. As template for the tandem mutant U-CR56D958N,D999N, pT7H6UbiFXCR56D958N was constructed first and then used as template in a second mutagenesis cycle. Mutagenesis primers were from DNA Technology A/S (Aarhus, Denmark). The initial melting temperature of all primers used for mutagenesis was designed to be at least 62 °C, and mutation sites were located in the central region of the primers. All constructs and mutations were verified by DNA sequencing using the Thermo Sequenase^TM II dye terminator cycle sequencing kit (Amersham Pharmacia Biotech).

The RAPd3 expression construct described by Ellgaard et al. (15) was modified to include three additional N-terminal residues (RAP residues 216-218) and an upstream methionine residue to allow removal of the N-terminal fusion partner by CNBr cleavage. Briefly, the extended RAPd3 construct was generated using the oligodeoxynucleotides 5'-GGC GGA TCC ATG GCT GAG TTC GAG GAG CC-3' and 5'-CAG CCA ACT CAG CTT CCT TTC GGG C-3' as primers and the pT7H6FX₂MRAP expression plasmid (33) as template in the PCR. The final expression vector, pT7H6UbiMetRAPd3, was obtained by inserting the BamHI-HindIII-digested PCR fragment into the pT7H6Ubi vector (15).

Protein Expression and Affinity Binding Analysis-- Recombinant RAP (33) and RAPd3 (15) were produced as described previously and were immobilized on Sepharose CL-6B (Amersham Pharmacia Biotech), which had been activated with 1,1'-carbonyldiimidazole (34). Recombinant U-CRxy protein was expressed in E. coli essentially as described (35), and the protein preparation was applied to a Ni²⁺-activated nitrilotriacetic acid-Sepharose column (36) and washed with 8 M urea, 500 mM NaCl, 50 mM Tris-HCl, pH 8.0, 10 mM 2-mercaptoethanol (buffer A) until stable A₂₈₀ of the effluent. The protein was eluted from the Ni²⁺-nitrilotriacetic acid-Sepharose with buffer A supplemented with EDTA to 10 mM and loaded to a Q-Sepharose column (Amersham Pharmacia Biotech) after desalting into 8 M urea, 10 mM NaCl, 50 mM sodium acetate, pH 5.0, 10 mM 2-mercaptoethanol. Elution was performed with a NaCl gradient, and when necessary the protein eluate was concentrated by ultrafiltration on a 10-kDa cut-off filter (Amicon). Refolding was carried out by dialysis against 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM CaCl₂, 3 mM reduced glutathione, and 0.3 mM oxidized glutathione for 24 h at 4 °C.

The mixtures of correctly and incorrectly folded U-CRxy fusion proteins were loaded on a RAPd3 column in MB buffer (140 mM NaCl, 10 mM HEPES, pH 7.4, 1 mM MgCl₂, 2 mM CaCl₂) supplemented with CaCl₂ to a final concentration of 12 mM, at 0.5 ml/min at 4 °C. Bound proteins were eluted with 100 mM glycine-HCl, pH 3.0, 150 mM NaCl, 20 mM EDTA. Non-RAP-binding and RAPd3-binding derivatives were loaded on a Superdex 75 (Prep grade) 10/30 column (Amersham Pharmacia Biotech), equilibrated in MB buffer and eluted at room temperature at a flow rate of 0.5 ml/min. Pure monomeric fractions eluted at peak position relative to a total column volume of 0.66 (~14.5 ml).

Where appropriate, the U-CRxy was gel-filtrated into 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM CaCl₂ and liberated from the ubiquitin fusion partner by cleavage with the endoproteinase FX_a using a 1:50 (w/w) enzyme/substrate ratio and an incubation time of 4-9 h at 4 °C. Uncleaved material, fusion partner, and factor X_a were removed by passage through Ni²⁺-nitrilotriacetic acid and factor X_a inhibitor Sepharoses (Protein Engineering Technology ApS, Aarhus, Denmark).

Protein Quantification and Examination of Structural Integrity of U-CRxy Derivatives-- Extinction coefficients (₂₈₀, M¹ cm¹) used for protein quantification were estimated using the ExPaZy server facilities (on the World Wide Web) to be 15380 (U-CR34, U-CR78), 14660 (U-CR45, U-CR56), 15940 (U-CR67), 13380 (U-CR89), and 8410 (U-CR910). The ₂₈₀ predicted for CR34 was verified by amino acid analysis. Aliquots of isolated protein were analyzed by nonreducing SDS-PAGE, followed by either Coomassie staining, where staining intensities for U-CRxy confirmed that the use of ₂₈₀ for the proteins was justifiable, or ⁴⁵Ca blotting, essentially as in Ref. 37. Briefly, identical samples were analyzed by SDS-PAGE or transferred to nitrocellulose membrane; washed three times with 10 mM Tris-HCl, pH 8.0, 60 mM KCl, 5 mM MgCl₂; and incubated for 10 min in the same buffer containing ⁴⁵CaCl₂ (1 mCi/liter) before washing with 50% ethanol, drying, and exposure for 24 h before analysis with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Disulfide Pattern Analysis: Generation of Peptides-- Approximately 250 µg of recombinant CR56 were solubilized in 5% formic acid and digested with pepsin using a 1:50 (w/w) enzyme/substrate ratio and an incubation time of 16 h. Following lyophilization, the digest was solubilized in 100 mM NH₄HCO₃, pH 8.0, and subjected to further proteolysis with trypsin using a 1:50 (w/w) enzyme/substrate ratio and an incubation time of 6 h. Finally, the peptide mixture was subjected to digestion with chymotrypsin using the same conditions as for trypsin. All digestions were at 37 °C. Following digestion and lyophilization, peptides were separated by reverse-phase high pressure liquid chromatography (HPLC) on a Vydac C₁₈ column using an Amersham Pharmacia Biotech system. The peptides were separated in 0.1% trifluoroacetic acid and eluted with a stepwise linear gradient of acetonitrile (B) developed over 50 min (0-5 min, 0% B; 5-40 min, 0-50% B; 40-50 min, 50-95% B) at a flow rate of 0.85 ml/min. The column was operated at 40 °C, and peptides were detected in the effluent by monitoring absorbance at 226 nm.

Sequence Analysis-- Edman degradation was performed on an Applied Biosystems 477A sequencer equipped with an on-line HPLC. For sample loading, isolated peptides (20-200 pmol) were pipetted onto polybrene-coated glass filters.

Mass Spectrometry-- Mass spectra were acquired with a Bruker BIFLEX matrix-assisted laser desorption/ionization time-of-flight instrument (Bruker-Franzen, Bremen, Germany) equipped with a 1-m flight tube, a reflector, a 337-nm nitrogen laser, and a 500-MHz digitizer. Thin film matrix surfaces were prepared using the fast evaporation technique (38) from -cyano-4-hydroxycinnamic acid (Sigma) dissolved in acetone/water (99:1) to 30 µg/µl. A 0.5-µl volume of analyte (0.1-10 pmol/µl) was deposited on the matrix surface and allowed to dry onto the crystals. Spectra were obtained by averaging 20-50 single-shot spectra and calibrated internally by co-crystallizing small amounts of angiotensin II (Sigma) and adrenocorticotropic hormone, fragment 18-39 (Sigma), with the analyte and by using the calibration constants of well known matrix ions.

Surface Plasmon Resonance (SPR) Analysis-- U-CRxy fusion proteins were immobilized on CM5 BIAcore sensor chips using the Amine Coupling Kit as described by the manufacturer (BIAcore, Sweden). After chip activation by the injection of 0.2 M N-ethyl-N-(3-dimethylaminopropyl)carbodiimide and 0.05 M N-hydroxysuccimide, purified U-CRxy proteins in 25 mM sodium acetate, pH 5.0, 150 mM NaCl, 10 mM CaCl₂ were diluted to a concentration of 10 µg/ml by the addition of 10 mM sodium acetate, pH 4.5, and passed through the BIAcore flow cell at a rate of 5 µl/min. After coupling of proteins, BIAcore chips were capped by exposure to 1 M ethanolamine, pH 8.5. Total protein coupling yields for U-CR34, U-CR56, and U-CR78 were 64/25, 64/47, and 57/17 fmol/mm² (Chip SM2310D1-98/SM2901E2-99), and for U-CR45, U-CR67, and U-CR89 yields were 33, 40, and 26 fmol/mm² (Chip SM2903E1-99), respectively. Analyte proteins were desalted into Ca-HBS buffer (HBS buffer with calcium added to 1.5 mM), and protein binding analysis was performed at a flow rate of 5 µl/min. Before loading of the protein sample, the chip was equilibrated in Ca-HBS buffer, which also was used as running buffer. Aliquots of 40 µl of protein sample were injected using the KINJECT option, and regeneration of the sensor chip was performed using 1.6 M glycine-HCl, pH 3.0. Kinetic parameters were determined by using the BIAevaluation program version 3.0 (BIAcore, Sweden).

U-CRxy Competition Analysis-- Labeling of RAP using the chloramine-T method (39) and ligand competition analysis (15) were essentially as described previously. Human LRP was purified from placenta essentially as described (40). Briefly, microtiter wells (Nunc Maxisorp, Denmark) were coated with approximately 1 µg/ml LRP by incubation for 16 h at 4 °C in 50 mM NaHCO₃, pH 9.6. After blocking with 5% bovine serum albumin for 2 h at room temperature, the wells were washed three times with MB buffer before incubation with ¹²⁵I-RAP (~7000 cpm/well) and 500 nM or 1 µM U-CRxy and derivatives (all proteins were in MB buffer) was performed for 16 h at 4 °C in MB buffer supplemented with 2% bovine serum albumin. Following washes with MB buffer, bound radioactivity was released by adding 10% SDS. Nonspecific binding of tracer to wells coated with LRP and inhibited with 10 mM EDTA was determined and subtracted from the values determined in the binding experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of a Minimal RAP Binding LRP Unit-- Fig. 1 outlines the structural architecture of LRP and the dissection of the second ligand binding cluster of complement-type repeats (cluster II) into single- or double-repeat fragments produced in E. coli cells. An identified high affinity RAP binding site in LRP (41) has previously been located to cluster II (42), but none of the tested single repeats (CR3, CR4, CR5, and CR6) displayed affinity for RAP or RAPd3 affinity columns after in vitro refolding. We subsequently generated a complete set of overlapping two-domain derivatives of cluster II, representing CR pairs CR34, CR45, CR56, CR67, CR78, CR89, and CR910. Except for the last construct, all bound the RAP and RAPd3 affinity matrices used for purification of the constructs.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Schematic representation of LRP and molecular dissection of the second cluster of complement-type repeats. Single-module and overlapping two-module fragments of cluster II were expressed as ubiquitin-fused proteins in an E. coli system.

Each RAP affinity-purified derivative was recovered as a pure homogeneous product, producing a well defined single band by SDS-PAGE analysis of nonreduced samples (Fig. 2A). Analysis of crude and affinity-purified receptor fragment preparations by nonreducing SDS-PAGE (Fig. 2B) showed that the well defined single bands corresponding to the affinity-purified species (U-CR34, U-CR45, U-CR56, U-CR67, U-CR78, and U-CR89) were abundant components in the crude refolding mixture. A similar prominent (i.e. probably correctly folded) component was also present in the crude U-CR910 refolding product, suggestive of efficient refolding of this receptor fragment also.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Tandem CR domain binding to RAP and RAP derivatives. A, generation of in vitro refolded mixtures of different folded species resulted in homogenous CR pair protein samples after RAP or RAP derivative chromatography. Lanes 1-6 represent U-CR34 (20.312 Da), U-CR45 (19.333 Da), U-CR56 (19.139 Da), U-CR67 (19.657 Da), U-CR78 (20.252 Da), and U-CR89 (19.914 Da) under nonreducing conditions. U-CR910 did not bind RAP. B, RAP affinity purification of U-CR56. Lane 1 shows a folding mixture of U-CR56, and lane 2 shows the nonbinding fraction after passage of a RAP affinity column. Lanes 3-6 represent native disulfide-linked U-CR56, purified by RAP affinity chromatography. Only the sharp band containing the correct folded protein (from lane 1) was retained on the column, whereas all incorrect folding products were not. Lane 7 represents a reduced (2-mercaptoethanol) sample of U-CR56, showing a decreased migration rate, indicating the presence of disulfide bridges in the RAP binding receptor fragment. Lane M, molecular mass values for marker proteins are 94, 67, 43, 30, 20.1, and 14.4 kDa (top to bottom).

In order to provide further evidence for correct folding we analyzed the disulfide-bridging pattern in one of the refolded, factor X_a-processed, and affinity-purified two-domain products, CR56. A set of disulfide-bridged peptides from digested constructs was isolated by reverse-phase HPLC, characterized by partial sequencing and mass spectrometry, and found compatible with the expected three Cys^I-Cys^III, Cys^II-Cys^V, and Cys^IV-Cys^VI disulfide bridges of both CR5 and CR6 (a data summary is presented in Table I).

To examine the ligand binding properties of affinity-purified two-repeat receptor fragments by SPR analysis, we prepared biosensor chips with immobilized fusion proteins U-CR34, U-CR45, U-CR56, U-CR67, U-CR78, and U-CR89 on the surface.

SPR analysis of the interaction of RAP and RAPd3 with immobilized two-repeat fragments showed that RAP (Fig. 3A) and RAPd3 (Fig. 3B) both bound strongly to U-CR34, U-CR45, U-CR56, U-CR67, U-CR78, and U-CR89. Experiments made with two separately prepared sensor chips coated with different surface densities of CR pairs produced virtually identical results. For RAP binding, fitting of the recorded sensorgrams to a simple one-site model generated estimated K_d values in the range of 1-5 nM for each two-repeat protein.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   SPR analysis of the binding of RAP and RAP domain 3 to array of LRP CR domain pairs. After purification of U-CR34, U-CR45, U-CR56, U-CR67, U-CR78, and U-CR89 by RAP affinity chromatography, they were immobilized on biosensor chips to estimate the affinity. Representative sensorgrams from the SPR binding analysis of RAP (5, 10, 20, 50, 100, 200, and 500 nM) (A) and RAP domain 3 (5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1µM, and 5 µM) (B) are shown, where plateau response levels in each sensorgram series correspond directly to ligand concentration. The estimated Kd for the binding of U-CR34, U-CR45, U-CR56, U-CR67, U-CR78, and U-CR89 to RAP were all in the range of 1-5 nM. Response units were normalized according to the amount of immobilized receptor fragment.

Given that U-CR34, U-CR45, U-CR56, U-CR67, U-CR78, and U-CR89 were all able to bind RAP when immobilized on biosensor chips, we conducted solid-phase competition analysis to examine whether they showed any difference in ability to compete with the binding between immobilized LRP and ¹²⁵I-RAP. As seen from the results shown in Fig. 4A, U-CR34, U-CR45, U-CR56, U-CR67, U-CR78, and U-CR89 were all able to compete the binding between native LRP and ¹²⁵I-RAP.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   CR-domain pair competition analysis of 125I-RAP binding to LRP. Inhibition of 125I-RAP binding to immobilized LRP by tandem CR modules from the second cluster of ligand binding repeats of LRP. The ordinate shows the ratio between bound and free 125I-RAP in wells relative to the ratio from wells with no added competitor (control). Plotted values represent mean values of pentaplicate measurements of two independent experiments, and vertical bars indicate the S.D. value. A, competition with U-CR34, U-CR45, U-CR56, U-CR67, U-CR78, and U-CR89 used at either 500 nM (first column) or 1 µM (second column) in each series. B, competition with U-CR34, U-CR45, U-CR56, and Asp  Asn mutant derivatives thereof (U-CR34D876N, U-CR34D917N, U-CR45D917N, U-CR45D958N, U-CR56D958N, U-CR56D999N, and U-CR56D958N,D999N) each at a concentration of 1 µM.

Tentative Identification from a Sequence Alignment of a Conserved Acidic Residue in LRP-- An alignment of the sequences of the eight repeats from LRP cluster II (CR3-10) and of those of the seven repeats of the ligand binding domain of LDLR (LB1-7) is shown in Fig. 5A. A striking difference is the conservation of a surface-exposed acidic residue located at the center position between Cys^IV and Cys^V. This acidic residue is conserved in most repeats of LRP cluster II, but only in few repeats of the LDLR domain. An acidic residue at this position was identified in the study by Rong et al. (Asp-23^LB4) as important for ligand binding (32). Notably, the only repeat in the LRP cluster that lacks the negative charged residue is CR10, which was the only repeat found here not to be involved in binding to RAP. Accordingly, we directed our experimental efforts toward investigations of the contribution of this conserved aspartic acid residue to high affinity RAP binding.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Identification of a conserved surface-exposed acidic residue. An alignment of CR3-CR10 (LRP) and LB1-LB7 (LDLR) is shown in A. The ~40-amino acid ligand binding repeats are aligned, resulting in six strictly conserved cysteines. Residues coordinating calcium via their side chain are marked with a downward arrow, and residues coordinating calcium via their backbone carbonyl are marked with a double downward arrow. At the position marked with an asterisk, an aspartic acid residue is conserved in 7(8) LRP modules, where CR10 is the only repeat not containing an acidic side chain. From the solved structure of CR8 (Protein Data Bank, accession code 1CR8; Ref. 30) the side chain of the conserved aspartic acid residue (Asp-1085CR8) is seen to be located at the molecular surface of the module and the backbone carbonyl oxygen coordinating calcium (B). The figure was generated using the Swiss PDB viewer.

Since U-CR34, U-CR45, and U-CR56 were slightly better inhibitors of RAP binding to LRP than either of U-CR67, U-CR78, and U-CR89 (Fig. 4A), we restricted the further analysis to these three fragments. The conserved aspartic acid residues in CR3 to CR6 (Asp-876, Asp-917, Asp-958, and Asp-999) were then replaced with asparagine residues to obtain U-CR34D876N, U-CR34D917N, U-CR45D917N, U-CR45D958N, U-CR56D958N, U-CR56D999N, and U-CR56D958N,D999N.

Isolation, Refolding, and RAP Binding of Asp-mutated Derivatives-- The expression levels of mutant proteins were virtually identical to those obtained for the wild-type two-domain fragments. However, the purification step employing RAP affinity chromatography, which was very efficient in isolating refolded U-CR34, U-CR45, and U-CR56, was less useful for the mutant proteins. U-CR34D876N, U-CR34D917N, U-CR56D958N, and U-CR56D999N exhibited decreased affinity for RAP and RAPd3 immobilized on Sepharose, and U-CR45D917N and U-CR45D958N did not bind at all. Instead of using RAP affinity chromatography, the U-CR45 mutant derivatives were purified by gel filtration. A data summary is given in Table II.

The decreased RAP affinity was apparently not a result of misfolding, since nonreducing SDS-PAGE analysis (Fig. 6A) revealed similar migration patterns for mutants and native two-domain proteins, suggesting the presence of fully oxidized mutant proteins with the authentic disulfide-bridge pattern. Furthermore, all mutant protein products were found to bind Ca²⁺ in 45-calcium blots with the same efficiency as the nonmutated protein (Fig. 6B). This strongly suggests that correct folding had been achieved, since calcium binding is dependent on a native conformation folding (27, 31). That calcium binding was not the result of one remaining functional site in the nonmutated module of a two-module protein was inferred from the results obtained for U-CR56D958N,D999N (Fig. 6B, lane 10). Here both conserved acidic residues (Asp-958 and Asp-999) in U-CR56 were replaced, each located at the critical position in the individual repeats, and calcium binding was found to be as strong as for native U-CR56. The U-CR45 derivatives showed weaker calcium binding relative to U-CR34 and U-CR56 (lanes 4-6 versus lanes 1-3 and 7-10). This was also true for wild-type U-CR45 and was therefore not indicative of improper folding of the mutant proteins.

View larger version (50K): [in this window] [in a new window]   Fig. 6.   SDS-PAGE analysis and 45CaCl2 binding of U-CR34, U-CR45, U-CR56, and mutant protein products. A, nonreducing SDS-PAGE analysis of aliquots of refolded protein either eluted from RAPd3 affinity chromatography (lanes 1-4, U-CR34, U-CR34D876N, U-CR34D917N, and U-CR45, respectively; lanes 7-9, U-CR56, U-CR56D958N, and U-CR56D999N, respectively) or after gel filtration on Superdex 75 column (lanes 5, 6 and 10, U-CR45D917N, U-CR45D958N, and U-CR56D958N,D999N, respectively). Lane 11, U-S15 (ubiquitin fused to the 15 N-terminal residues of human RNase A); lane M, molecular mass values for marker proteins are 94, 67, 43, 30, 20.1, and 14.4 kDa (from top to bottom, although reduced). The similar migration pattern for Asp  Asn derivatives and wild-type U-CRxy suggests an equal disulfide pattern in mutant as in native CR-domain fragments. B, similar protein samples as in A were transferred to nitrocellulose membranes and blotted with 45CaCl2 to further test for the correct folding of the mutant proteins. The arrows indicate identical positions in A and B. The missing band in lane 11 indicates that calcium binding is not a result of the ubiquitin fusion partner.

The decreased RAP affinity was further confirmed by solid-state competition analysis of the mutant derivatives (Fig. 4B). When 1 µM U-CR34 was used as competitor, 33% of added ¹²⁵I-RAP bound to immobilized LRP as compared with wells without any competitor. In contrast, 55 or 85% binding was achieved in the presence of 1 µM U-CR34D876N or U-CR34D917N, respectively. A similar effect was observed for U-CR45 and U-CR56 where RAP binding to LRP was also inhibited less efficiently by Asp  Asn mutant proteins. The data are summarized in Table II.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study defines two complement-type repeats as the minimal unit of LRP required for the high affinity binding to RAP. Instead of only one site responsible for RAP recognition, as previously suggested (43, 44), we demonstrate multisite binding of RAP to LRP cluster II. Furthermore, simultaneous binding of RAP to two adjacent CR modules is suggested, since impaired RAP binding is not located to only one repeat but is dependent of residues in both repeats in the two-repeat proteins.

From these results, it is tempting to suggest a model of RAP binding to members of the LDLR family, where binding is mediated by the interaction between one RAP domain and one two-CR unit. This model implies the existence of a high number of binding sites on LRP in agreement with previous reports (20). Furthermore, because RAP contains at least two high affinity domains, RAP may cross-link regions of LRP in various ways. Fig. 7 is a putative model of the complex binding between RAP and LRP. The presence of multiple binding sites provides an explanation of the unique antagonizing effect of RAP and also explains the fact that ₂M* affinity chromatographic purification of LRP with associated RAP is possible, despite RAP being an inhibitor of ₂M* binding.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Putative model of LRP multisite RAP binding. The identification of multiple RAP binding sites, presented by only two complement-type repeats, suggests the possible binding of multiple RAP molecules to each LRP molecule. This gives a plausible explanation of why RAP is co-purifying with LRP when RAP affinity chromatography is used (I). We have previously shown that in addition to RAPd3, also RAP domains 1 and 2 bind RAP, although with weaker affinity than RAPd3. This gives RAP the ability to cross-link repeats from different clusters, resulting in a possible conformational change (II). Note that RAP is shown as a three-domain protein, but an alternative four-domain architecture has also been suggested (17).

We also describe a preference for an acidic residue in the second cluster of ligand binding modules from LRP, identified by the alignment of the repeats with those from LDLR. The acidic residue is not buried inside the module as are other acidic residues, previously suggested to be involved in ligand recognition but now known to coordinate calcium binding. The present work considers only residues Asp-876, Asp-917, Asp-958, and Asp-999 present in the four amino-terminal repeats (CR3-CR6) of cluster II, but in view of the equivalent binding of RAP also to tandem repeats comprising CR7, CR8, and CR9, we predict a similar role of the conserved aspartic acid residues in these repeats (Asp-1037, Asp-1085, and Asp-1128).

According to an alignment of sequences of repeats from other human members of the LDLR family (not shown) a negative charged residue at the center position between Cys^IV and Cys^V is present at a high frequency in other RAP binding receptors as well. It is therefore tempting to speculate whether the number of modules containing the conserved acidic residue correlates with efficient binding of RAP in general. In fact, such a tendency seems apparent when comparing the RAP binding properties of various LDLR-like receptors. A schematic representation of the LDLR family is shown in Fig. 8.

View larger version (35K): [in this window] [in a new window]   Fig. 8.   Schematic representation of the putative RAP-binding acidic residues in various repeats from the human low density lipoprotein receptor family. Distribution of acidic residues in clusters of ligand binding repeats from members of the human LDLR family is shown. D and E represent aspartic acid and glutamic acid residues, respectively, at the position shown by an asterisk in Fig. 5, obtained from alignment of sequences of all of the repeats under the same conditions as employed in the alignment shown in Fig. 5. Triangles represent modules not present in the major splice variant of VLDLR (CR3) and ApoER2 (CR4-6) in the brain. Removing these modules containing an acidic residue has been reported to decrease the affinity for RAP (47, 50). RAP binding has been observed for ApoER2, LDLR, and SorLA-1 as well as for constructs comprising CR3-6, CR7-10, CR16-20, CR21-26, and CR27-31 of LRP and CR1-3, CR1-5, and CR1-8 of VLDLR.

Obermoeller et al. (20) demonstrated efficient RAP binding of the second and the fourth cluster and low affinity binding to the third cluster, whereas no binding was observed to the first cluster of CR modules from the LRP. This observation is in good correlation with the number of acidic residues being 1(2), 7(8), 4(10), and 8(11)³ (see Fig. 8). Further detailed investigation showed that whereas both the amino- and the carboxyl-terminal halves of cluster II and cluster IV were able to bind immobilized RAP on nitrocellulose membranes, only the carboxyl-terminal part of cluster III showed affinity for RAP (20). This also supports the present hypothesis, since only one repeat in the amino-terminal part contains an acidic residue at the position in contrast to 3(5) repeats in the carboxyl-terminal end.

Furthermore, only low affinity interaction between RAP and LDLR was reported by Medh et al. (45), in agreement with 3(7) CR modules in the LDLR ligand binding domain harboring a negative charge at the center position between Cys^IV and Cys^V.

Finally, the VLDLR, apoER2, and SorLA-1, having 6(8), 6(7), and 5(11) Asp/Glu CR modules, respectively, bind strongly to RAP (46-48). It should be noted that various splice variants of both apoER2 and VLDLR have been identified (47, 49), and the reported RAP binding properties of those show no discrepancies with our hypothesis. The major variant of apoER2 in human brain lacking CR4-6 of the normally seven repeats present shows efficient RAP recognition (47), where the remaining four repeats all contain the negatively charged residue. The binding was found to be weaker than for the seven-repeat protein, in agreement with the loss of two repeats containing Asp.

Several groups have carried out detailed analysis of the RAP binding properties of VLDLR. Savonen et al. (21) reported that RAP interacts as well with CR1-3 as with CR1-5, but not with CR6-8. These results are in agreement with another study by Rettenberger et al. (50) demonstrating impaired RAP binding to a naturally occurring VLDLR variant lacking the third CR module, which contains a negatively charged residue. An analysis by Mikhailenko et al. (51) suggested that the RAP binding site of VLDLR is located within the amino-terminal four class A repeats and also suggested that the most amino-terminal CR is especially important for RAP binding.

It should be noted that the ligand binding repeat also is present in several proteins not exhibiting other homologies with the LDLR family. In these proteins, there is no preference for an acidic residue (e.g. the two novel discovered serine proteases, Matriptase (52), containing four modules with a tryptophan, a valine, and two lysine residues at the critical position, and TMPRSS2 (53), with a single class A repeat with a valine residue). No RAP binding has been reported for these molecules.

In conclusion, the present molecular dissection of LRP cluster II has resulted in the identification of a range of different double CR domains as independent RAP-binding units. This implies a multiplicity of RAP binding sites in LRP. The presence of a partly conserved acidic residue among clusters of CR repeats is reported to enhance RAP affinity, but solving the structure of CR pairs and RAP domains will provide further information on the molecular interactions.

	ACKNOWLEDGEMENTS

We thank Ove Lillelund for excellent technical assistance and Dr. J. Herz for LRP cDNA.

	FOOTNOTES

^* The present work was supported by Danish Biotechnology Program Grant 9502045.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 45 86 20 20 00; Fax: 45 86 18 01 85; E-mail: hct@biobase.dk.

Published, JBC Papers in Press, March 24, 2000, DOI 10.1074/jbc.M000507200

² Roman numerals designate relative sequence positions of conserved cysteine residues within the CR domain.

³ In the nomenclature n(m), n represents the number of repeats containing an acidic residue, and m represents the total number of repeats in the cluster.

	ABBREVIATIONS

The abbreviations used are: LDLR, low density lipoprotein receptor; VLDLR, very low density lipoprotein receptor; RAP, receptor-associated protein; LRP, ₂-macroglobulin receptor/LDLR-related protein; CR, LRP complement-type repeat; apoER2, apolipoprotein E receptor 2; RAPd3, RAP residues 216-323; ₂M*, transformed ₂-macroglobulin; LB, LDLR ligand binding repeat; PCR, polymerase chain reaction; U-CRxy, fusion protein containing ubiquitin and complement-type repeats x and y; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; SPR, surface plasmon resonance; HBS, Hepes-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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