Three Complement-like Repeats Compose the Complete α2-Macroglobulin Binding Site in the Second Ligand Binding Cluster of the Low Density Lipoprotein Receptor-related Protein

Given the importance of the low density lipoprotein receptor-related protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of α2-macroglobulin (α2M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity α2M binding to the eight complement repeat (CR)-containing cluster 2 of LRP. In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for α2M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (Kd = 130 nm) the same as for isolated RBD to intact LRP. This Kd is 30-fold smaller than for RBD to CR5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster. Given the importance of the low density lipoprotein receptor-related protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of α2-macroglobulin (α2M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity α2M binding to the eight complement repeat (CR)-containing cluster 2 of LRP. In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for α2M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (Kd = 130 nm) the same as for isolated RBD to intact LRP. This Kd is 30-fold smaller than for RBD to CR5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster. The low density lipoprotein receptor-related protein (LRP) 2The abbreviations used are: LRP, low density lipoprotein receptor-related protein; α2M, α2-macroglobulin; CR, complement-like repeat; GSH, reduced glutathione; GSSG, oxidized glutathione; LDL, low density lipoprotein; LDLR, LDL receptor; RAP, receptor-associated protein; RBD, receptor binding domain; TEV, tobacco etch virus NIa protease; GdnHCl, guanidine HCl; Ni-NTA, nickel-nitrilotriaceticacid;β-ME,β-mercaptoethanol;HPLC, high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid. is an essential member (1Herz J. Clouthier D.E. Hammer R.E. Cell. 1992; 71: 411-421Abstract Full Text PDF PubMed Scopus (511) Google Scholar) of the low density lipoprotein (LDL) family of mosaic receptor proteins that are responsible for binding and internalization of a large number of protein ligands (2Herz J. Strickland D.K. J. Clin. Investig. 2001; 108: 779-784Crossref PubMed Scopus (897) Google Scholar). Each of these receptors contains one or more clusters of ligand binding domains, known both as complement-like repeats (CR) and LDL receptor type A modules (3Catterall C.F. Lyons A. Sim R.B. Day A.J. Harris T.J. Biochem. J. 1987; 242: 849-856Crossref PubMed Scopus (84) Google Scholar). Whereas LDLR contains only one such cluster of 7 CR domains, LRP has four clusters containing 2, 8, 10, and 11 repeats, counting from the N terminus (4Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Crossref PubMed Scopus (742) Google Scholar, 5Kristensen T. Moestrup S.K. Gliemann J. Bendtsen L. Sand O. Sottrup-Jensen L. FEBS Lett. 1990; 276: 151-155Crossref PubMed Scopus (256) Google Scholar, 6Strickland D.K. Ashcom J.D. Williams S. Burgess W.H. Migliorini M. Argraves W.S. J. Biol. Chem. 1990; 265: 17401-17404Abstract Full Text PDF PubMed Google Scholar). Commonly used nomenclature, thus, describes cluster 2 as being composed of domains CR3 through CR10. Each CR domain contains three conserved disulfide bridges and a functionally required calcium binding site within 40-42 residues. These domains are linked by variable length, flexible linkers that are likely to afford considerable orientational freedom to each CR domain with respect to others in the cluster (7Beglova N. North C.L. Blacklow S.C. Biochemistry. 2001; 40: 2808-2815Crossref PubMed Scopus (42) Google Scholar). Although the cysteines and calcium-coordinating residues are highly conserved, most of the remaining ∼30 residues are variable (8Simonovic M. Dolmer K. Huang W. Strickland D.K. Volz K. Gettins P.G.W. Biochemistry. 2001; 40: 15127-15134Crossref PubMed Scopus (54) Google Scholar). This potentially gives LRP a wide variety of ligand recognition sites within its four clusters of 31 CR domains and may in part explain the very much broader range of ligands that can bind and be internalized by LRP compared with the much simpler LDLR. Thus, LRP binds ligands as diverse as ∼760-kDa α2M-protease complexes, various ∼100-kDa serpin-proteinase complexes such as plasminogen activator inhibitor 1-urokinase-like plasminogen activator (uPA), thrombin-antithrombin, and protease nexin-1-uPA, apolipoprotein E, and lipoprotein lipase, amyloid precursor protein, complement C3, and lactoferrin (2Herz J. Strickland D.K. J. Clin. Investig. 2001; 108: 779-784Crossref PubMed Scopus (897) Google Scholar). One of the best studied LRP ligands is α2M. α2Misan abundant, homo-tetrameric pan-proteinase inhibitor (9Sottrup-Jensen L. J. Biol. Chem. 1989; 264: 11539-11542Abstract Full Text PDF PubMed Google Scholar) that is also reported to bind various growth factors (10Crookston K.P. Webb D.J. LaMarre J. Gonias S.L. Biochem. J. 1993; 293: 443-450Crossref PubMed Scopus (67) Google Scholar) and that is evolutionarily related to the complement proteins C3, C4, and C5 (11Sottrup-Jensen L. Stepanik T.M. Kristensen T. Lqnblad P.B. Jones C.M. Wierzbicki D.M. Magnusson S. Domdey P.B. Wetsel R.A. Lundwall Å. Tack B.F. Fey G.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 9-13Crossref PubMed Scopus (138) Google Scholar). Binding of α2M to LRP requires the exposure of a previously hidden receptor binding domain (RBD) that constitutes the C-terminal 138 residues of each α2M chain. Exposure of RBD results from the conformational transformation that occurs upon complex formation with proteinase. LRP can, thus, discriminate strongly between native and activated α2M species. Internalization of complexed α2M can result not only in internalization and degradation of the complexed proteinase but also in intracellular signaling responses, making this ligand an especially important one to study (12Lutz C. Nimpf J. Jenny M. Boecklinger K. Enzinger C. Utermann G. Baier-Bitterlich G. Baier G. J. Biol. Chem. 2002; 277: 43143-43151Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). It is thought that the RBD of α2M, whose structure is known and consists of a β-sandwich edged by a single α-helix (13Huang W. Dolmer K. Liao X. Gettins P.G.W. J. Biol. Chem. 2000; 275: 1089-1094Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), is solely responsible for binding of α2M-proteinase complexes to LRP (5Kristensen T. Moestrup S.K. Gliemann J. Bendtsen L. Sand O. Sottrup-Jensen L. FEBS Lett. 1990; 276: 151-155Crossref PubMed Scopus (256) Google Scholar, 6Strickland D.K. Ashcom J.D. Williams S. Burgess W.H. Migliorini M. Argraves W.S. J. Biol. Chem. 1990; 265: 17401-17404Abstract Full Text PDF PubMed Google Scholar, 14Van Leuven F. Marynen P. Cassiman J.-J. van den Berghe H. Biochem. J. 1982; 203: 405-411Crossref PubMed Scopus (52) Google Scholar, 15Sottrup-Jensen L. Gliemann J. Van Leuven F. FEBS Lett. 1986; 205: 20-24Crossref PubMed Scopus (87) Google Scholar). Nevertheless, although intact α2M can bind to LRP with an affinity that reaches subnanomolar at high LRP density, isolated RBD binds with a Kd of 100-200 nm (15Sottrup-Jensen L. Gliemann J. Van Leuven F. FEBS Lett. 1986; 205: 20-24Crossref PubMed Scopus (87) Google Scholar, 16Enghild J.J. Thøgersen I.B. Roche P.A. Pizzo S.V. Biochemistry. 1989; 28: 1406-1412Crossref PubMed Scopus (59) Google Scholar). The difference is thought to be due to the tetrameric nature of α2M allowing interaction with two or more LRP molecules when LRP is present at high density or to an interaction with more than one cluster of CR domains within a single LRP molecule. Thus, it has been reported that α2M can bind to both cluster 2 and cluster 4 when each cluster is expressed as a minreceptor (17Neels J.G. van den Berg B.M.M. Lookene A. Olivecrona G. Pannekoek H.P. van Zonneveld A.-J. J. Biol. Chem. 1999; 274: 31305-31311Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Given the importance both of LRP as an essential endocytosis and signaling receptor and of α2M-proteinase complexes as an LRP ligand, an understanding of the specificity of their interaction is a very worthwhile goal. An excellent starting point is the known importance of RBD in binding, the identification of two lysine residues within RBD that are required for tight binding to LRP (18Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), the localization of high affinity α2M binding to CR cluster 2 of LRP (17Neels J.G. van den Berg B.M.M. Lookene A. Olivecrona G. Pannekoek H.P. van Zonneveld A.-J. J. Biol. Chem. 1999; 274: 31305-31311Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and our earlier observation that RBD can bind to CR3, the first CR domain of cluster 2, with modest affinity (19Dolmer K. Huang W. Gettins P.G.W. J. Biol. Chem. 2000; 275: 3264-3271Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In the present study we have built on this and attempted to identify the minimum additional CR domains that together with CR3, might constitute the full binding site for RBD and, hence, α2M on LRP. We report on the ability of the triple domain construct of CR3-4-5 to bind RBD with an affinity the same as for isolated RBD to intact LRP and to do so in a manner that requires the identified critical lysine residues, although in a manner that is almost pH-independent within the range of pH values encountered between extracellular and internal compartment environments. The latter finding supports the recently proposed model of intracellular ligand dissociation that involves displacement by YWTD domains adjacent to the CR-containing ligand binding clusters resulting from a pH-dependent increase in affinity of the competing YWTD domain for the CR cluster (20Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Science. 2002; 298: 2353-2358Crossref PubMed Scopus (385) Google Scholar). Expression, Purification, and Refolding of Wild Type and Variant Human RBDs—DNA encoding 1304SLKXnGNA1451 of human α2M was cloned into pET-15b (Novagen) as previously described (21Huang W. Dolmer K. Liao X.B. Gettins P.G.W. Protein Sci. 1998; 7: 2602-2612Crossref PubMed Scopus (27) Google Scholar). pET-15b-RBD was transformed into BL21-(DE3), grown in 2YT medium to A600 = 0.6-0.8, and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside. Cells were harvested after 4-5 h. Inclusion bodies were isolated after sonication and dissolved in 6 m GdnHCl, 100 mm NaPi, pH 7.4. RBD was loaded on a Ni-NTA Superflow (Qiagen) column, washed with 6 m GdnHCl, 100 mm NaPi, pH 6.0, and eluted with 6 m GdnHCl, 100 mm sodium acetate, pH 4.5. All buffers were degassed, and 0.1% (vol) β-ME was added just before use. For efficient refolding of RBD, an on-column method based on Oganesyan et al. (22Oganesyan N. Kim S.-H. Kim R. J. Struct. Funct. Genomics. 2005; 6: 177-182Crossref PubMed Scopus (38) Google Scholar), was used. The pH of an aliquot of purified RBD was adjusted to 7.4 by the addition of 2 m Tris base and diluted to 100 ml with 6 m GdnHCl, 100 mm NaPi, 0.1% β-ME, pH 7.4. RBD was bound to 70 ml of Ni-NTA Superflow in a batch at 1 mg of protein/ml of resin and packed in a column. Upon washing with 3 column volumes of 6 m urea, 100 mm NaPi, 0.1% β-ME, pH 7.4, the column was washed with 500 ml of 0.1% SDS, 0.1% β-ME, in PBS500 (20 mm NaPi, 500 mm NaCl, pH 7.4) and refolded with 500 ml of 5 mm β-cyclodextran, 0.1% β-ME, and 0.1% 2-hydroxyethyldisulfide in PBS500 overnight. After washing with 2 column volumes of 50 mm NaPi, 300 mm NaCl, 10 mm imidazole, pH 7.4, refolded RBD was eluted with 50 mm NaPi, 300 mm NaCl, 250 mm imidazole, pH 7.4. Unfolded RBD remained on the column and, after a wash with two column volumes of PBS buffer to remove imidazole, could be subjected to a second round of refolding starting with the wash with 6 m urea buffer as above. Almost all RBD loaded on the column could be refolded in 3-4 cycles. After dialysis against 100 mm sodium acetate, pH 4.5, RBD was cleaved with 1/100 (weight) papain for 2 h at room temperature to remove the His tag. The resulting domain is the one used for previous structural studies (13Huang W. Dolmer K. Liao X. Gettins P.G.W. J. Biol. Chem. 2000; 275: 1089-1094Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and is also the domain that is produced by papain cleavage of activated plasma α2M. Iodoacetamide was added to 5 mm, and the pH was adjusted to 8.0 with 2 m Tris base to inhibit papain. The His tag-cleaved RBD was dialyzed against 20 mm Tris-HCl, 50 mm NaCl, pH 8.0. The final purification step was performed on a Q-Sepharose HP column, equilibrated in 20 mm Tris-HCl, pH 8.0, and eluted with a gradient of 50-500 mm NaCl. A plasmid carrying K1370M/K1374M RBD was prepared from pET-15b-RBD by a single QuikChange (Stratagene) reaction. Expression, purification, and refolding were identical to those of wild type RBD. Expression, Purification, and Refolding of Complement Repeat Fragments—DNA encoding CR3-4 (853QCQXnCSA933) of human LRP was cloned into pGEX-2T (Amersham Biosciences) modified to contain a TEV proteinase cleavage site. BL21 cells containing pGEX-CR3-4 were grown in 2YT medium to an A600 = 0.8 at 37 °C. Expression was induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside, and the temperature was lowered to 25 °C. Cells were harvested after 5-6 h. The glutathione S-transferase fusion protein was purified as previously described (23Dolmer K. Huang W. Gettins P.G.W. Biochemistry. 1998; 37: 17016-17023Crossref PubMed Scopus (29) Google Scholar), cleaved with TEV protease, and repurified on GSH-Sepharose. CR3-4, collected from the flow-through, was further purified by reverse phase HPLC on a Vydac C18 column (10 × 250 mm) eluted with a gradient of 9-54% acetonitrile in 0.1% trifluoroacetic acid. Peak fractions were pooled and lyophilized. DNA encoding CR3-4-5 (853QCQXnCAY973) of human LRP was cloned into pQE30 (Qiagen) modified to contain a protein G fusion protein and a TEV site. The CR3-4-5 fusion protein was expressed in SG13009 pREP4 cells (Qiagen), grown to an A600 = 0.6 at 37 °C in 2YT medium, and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside. Cells were harvested after 4-5 h. The His6protG-CR3-4-5 fusion protein was purified on a Ni-NTA Superflow column according to the manufacturer's protocol (QiaExpressionist). After cleavage with TEV, the mixture was passed over the Ni-NTA column, and the flow-through was further purified by reverse phase HPLC as described above for CR3-4. DNA encoding CR5-6-7 (934RTC XnCTN1053) was cloned into pQE30 (Qiagen) modified to contain a TEV proteinase cleavage site. His6-tagged CR5-6-7 was expressed, purified, and processed essentially as described above for CR3-4-5 to give CR5-6-7 without a His tag. Purified CR3-4, CR3-4-5, and CR5-6-7 were refolded using a modification of the previously described protocol (23Dolmer K. Huang W. Gettins P.G.W. Biochemistry. 1998; 37: 17016-17023Crossref PubMed Scopus (29) Google Scholar). Each protein was dissolved in a minimal volume of 6 m GdnHCl, 50 mm Tris-HCl, pH 8.5, and reduced with a 2× molar excess (over Cys) of dithiothreitol for 30 min. Each fragment was then diluted with refolding buffer (50 mm Tris-HCl, 10 mm CaCl2, pH 8.5) and mixed with a 50% molar excess of glutathione S-transferase-receptor-associated protein (RAP) for a final concentration of complement repeat fragment of 0.1 mg/ml. The refolding mixture was dialyzed at room temperature against 4 liters of degassed refolding buffer containing 1 mm GSH and 0.5 mm GSSG for 24 h with N2 bubbling to avoid oxidation by O2 followed by 24 h of refolding at 4 °C without bubbling. To purify correctly folded complement repeat fragments, the mixture was dialyzed against 3 × 4 liters of 20 mm Tris-HCl, 50 mm NaCl, 10 mm CaCl2, pH 8.0, and loaded on a GSH-Sepharose column equilibrated in the same buffer. After a brief wash to remove unbound material, folded fragment bound to glutathione S-transferase-RAP was eluted with a buffer containing 20 mm Tris-HCl, 50 mm NaCl, 4 mm EDTA, pH 8.0. LRP fragments were further purified by reverse phase HPLC as described above. In each case, the folded protein eluted faster than the reduced protein before refolding. Folded CR3-4-5 and CR5-6-7 were dialyzed against 20 mm Tris-HCl, 50 mm NaCl, 0.1 mm CaCl2, pH 8.0, and loaded onto a Q-Sepharose-HP column (10 × 100 mm). The protein was eluted with a gradient of 50-1000 mm NaCl. As a final purification step to ensure that only correctly disulfide-bonded and, hence, functional protein was present, folded CR3-4-5 was mixed with His6-RBD in the presence of 1 mm CaCl2 and passed over a small Ni-NTA column. After washing with 5 column volumes of buffer, CR3-4-5 capable of binding RBD was eluted with a buffer containing 10 mm citrate. Traces of His6-RBD were removed by ion exchange chromatography on a Q-Sepharose HP column, eluted with a gradient of 0-1 m NaCl in 20 mm Tris-HCl, 1 mm EDTA, pH 8.0. Protein Concentrations—For each protein the extinction coefficient was calculated using the formula ϵ280 nm (m-1 cm-1) = 5500 × (number of tryptophans) + 1490 × (number of tyrosines) + 120 × (number of disulfide bridges). This empirical formula has been shown to be accurate to within a few percent of experimentally determined extinction coefficients for most proteins (24Pace C.N. Vajdos F. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar). Fluorescence Spectroscopy—Binding experiments were performed on a PTI Quantamaster instrument equipped with double monochromators on both the excitation and emission sides. Because RBD does not contain tryptophan residues, the tryptophan residues in CR3-4(-5) and CR5-6-7 could be selectively excited at 295 nm and RBD binding followed by the increase in tryptophan fluorescence at 325 nm. The experiments were performed in 20 mm Tris-HCl, 150 mm NaCl, 5 mm CaCl2, pH 7.4, containing 0.1% PEG 20,000 to avoid protein adsorption to the acrylic cuvette. Low pH binding experiments were performed in similar buffers containing 20 mm Bis-Tris (pH 6.5) or 20 mm MES (pH 6.0 and 5.5). Spectra of the apo-forms of CR3, CR3-4, CR3-4-5, and CR5-6-7, were obtained in 20 mm Tris-HCl, 150 mm NaCl, 10 μm EDTA, pH 7.4, containing 0.1% polyethylene glycol 20,000. Determination of Kd Values—Dissociation constants were determined by fitting fluorescence titration data to a single binding site model by non-linear least-squares fitting using the program Scientist (MicroMath, Salt Lake City, UT). It should be noted that LRP fragment concentrations used (0.5 μm) were above the Kd for the tightest binding fragment CR3-4-5, so that the condition for 50% saturation to occur at a concentration of titrant equal to the Kd is not met. SDS and Non-denaturing PAGE—SDS-PAGE was performed using a Novex 10-20% gel (Invitrogen) and standard Laemmli buffers. Non-denaturing PAGE was performed using a Novex 8% gel prerun for 4 h at 15 mA in 375 mm Tris-HCl, 5 mm CaCl2, pH 8.8, to introduce Ca2+ to the gel. The gel was run in the standard Tris-glycine buffer system supplemented with 5 mm CaCl2 at 15 mA until the dye front reached the bottom of the gel. Characterization of CR3-4, CR3-4-5, and CR5-6-7—We have previously shown that the first complement-like repeat in the second cluster of LRP, CR3, binds RBD weakly, although with sufficient affinity (Kd ∼ 140 μm) to suggest that it forms part of the RBD binding site on LRP (19Dolmer K. Huang W. Gettins P.G.W. J. Biol. Chem. 2000; 275: 3264-3271Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We therefore expressed first CR3-4 and then CR3-4-5 to determine whether the full affinity of RBD for LRP could be reproduced by adding contiguous domains and to enable evaluation of the contributions of individual domains to the overall binding energy. Because each CR domain contains three disulfide bonds, all of which are critical for the correct fold and, hence, function in ligand binding of the domain (25Blacklow S.C. Kim P.S. Nat. Struct. Biol. 1996; 3: 758-761Crossref PubMed Scopus (108) Google Scholar), we took great care to ensure that only correctly folded material was used in subsequent binding studies. This was a particular concern since initial preparations using the previously described method for refolding CR3-4 gave only a small fraction that appeared to be folded, as judged by the shift in mobility in reverse phase HPLC upon reduction. We, therefore, modified the published protocol to include the chaperone RAP, shown to be important for LRP expression and function in vivo (26Willnow T.E. Armstrong S.A. Hammer R.E. Herz J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4537-4541Crossref PubMed Scopus (247) Google Scholar, 27Bu G. Rennke S. J. Biol. Chem. 1996; 271: 22218-22224Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). To aid in the subsequent purification of correctly folded CR3-4, we used glutathione S-transferase-tagged RAP, which after dialysis of the folding mixture to remove GSH, bound GSH-Sepharose with the folded CR3-4 still bound. Folded CR3-4 was then eluted with EDTA. In the case of CR3-4-5, with nine disulfides, an extra affinity purification step using RBD was used to eliminate any species that did not bind with the highest affinity. SDS-PAGE showed that under both non-reducing and reducing conditions, the resulting CR3-4 and CR3-4-5 gave only single bands, suggesting that preparations were homogeneous (Fig. 1). In contrast, preparations of CR3-4-5 that had not undergone the additional RBD affinity purification step showed the presence of additional bands under non-reducing conditions (not shown). Calcium binding to the complement repeats is also indicative of correct folding and can be followed by the increase in tryptophan fluorescence. CR3, CR3-4, and CR3-4-5 fluorescence in the absence and presence of saturating Ca2+ shows a marked increase due to Ca2+ binding (Fig. 2). Stoichiometric titrations with Ca2+ confirmed the ability of each domain to bind Ca2+ (not shown). The variation in fluorescence enhancement reflects the different properties of each domain, which has been seen elsewhere for CR domains from both LRP and LDLR (8Simonovic M. Dolmer K. Huang W. Strickland D.K. Volz K. Gettins P.G.W. Biochemistry. 2001; 40: 15127-15134Crossref PubMed Scopus (54) Google Scholar, 23Dolmer K. Huang W. Gettins P.G.W. Biochemistry. 1998; 37: 17016-17023Crossref PubMed Scopus (29) Google Scholar) and perhaps also a degree of internal quenching that makes the fluorescence intensities of multidomain constructs less than the sum of contributions from the isolated constituent domains.FIGURE 2Fluorescence spectra of CR3, CR3-4, CR3-4-5, and CR5-6-7 in the absence (lower trace) and presence of 1 mm calcium (upper trace). All spectra are normalized to that of CR3 without calcium.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For subsequent use as an important negative control for RBD binding, we also expressed and characterized CR5-6-7. As with the above fragments, only a single band was obtained on SDS-PAGE under reducing or non-reducing conditions (data not shown). Ca2+ binding showed a comparable large increase in tryptophan fluorescence (Fig. 2), confirming correct folding of domains. RBD Binding to CR3-4 and CR3-4-5 by Gel Shift—As a first, qualitative measure of the affinities of CR3-4 and CR3-4-5 for RBD, non-denaturing PAGE was used. RBD and the two CR constructs alone showed large differences in mobility. No shift was seen for a 1:1 mixture of CR3-4 and RBD, suggesting that the interaction was relatively weak (Fig. 3, lanes 1, 3, and 4). For CR3-4-5, however, there was not only a complete shift for the two component proteins, but the resulting complex had higher mobility than either component (Fig. 3, lanes 1, 5, and 6). This might indicate that CR3-4-5 is transformed from an extended conformation in the absence of RBD to a compact conformation when wrapped around the more globular RBD. RBD Binding to CR3-4, CR3-4-5, and CR5-6-7 by Fluorescence-monitored Titration—RBD contains 6 tyrosines, but no tryptophans, whereas each CR domain contains one tryptophan. When complexed with RBD, neither CR3-4, CR3-4-5, nor the control CR5-6-7 shows a large change in the intensity of the tryptophan fluorescence, although there is a blue shift of 5-6 nm of the emission peak (Fig. 4). We were, therefore, able to follow RBD binding to each of these LRP fragments by selectively exciting the single tryptophan residue in each complement-like repeat at 295 nm and monitoring tryptophan emission at 325 nm where the change in intensity is largest. For all three fragments, CR3-4, CR3-4-5, and CR5-6-7, titration curves for RBD binding were saturable and could be fitted to a single binding site (Fig. 5), confirming the homogeneity of the preparations. Whereas CR3 alone had previously been found to bind to RBD with a Kd of only 140 μm, CR3-4 bound to RBD much tighter, with a Kd of 1.5 μm, and CR3-4-5 bound tighter still, with a Kd more than 1 order of magnitude smaller (130 nm) (Table 1). The significance of the latter Kd is that it matches the values reported for binding of RBD to intact LRP (100-200 nm) and so implies that CR3-4-5 constitutes the whole of the RBD binding site within LRP. In contrast, whereas CR5-6-7 still bound in a saturable manner, with a stoichiometry of 1:1, its affinity for RBD (4 μm, Table 1) was lower than for the tandem construct CR3-4, demonstrating the strong preference for RBD binding at CR3-4-5 rather than at other possible sites, with a 30-fold lower Kd for binding at CR3-4-5 than at CR5-6-7.FIGURE 5Titration curves for RBD binding to LRP fragments. CR3-4 (top left), CR3-4-5 (top right), CR5-6-7 (bottom right). Bottom left, K1370M/K1374M RBD binding to CR3-4-5. The LRP fragment concentration was 0.5 μm in each case. Note that this is higher than the Kd for CR3-4-5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Affinity of RBD and α2M for CR fragments and LRPProteinsRBD:CR3aFrom NMR titration (19).RBD:CR34RBD:CR3-4-5K-M:CR3-4-5RBD:CR5-6-7RBD:LRPbFrom Refs. 15 and 16.α2M:LRPcFrom single/multiple receptor model (28, 32).Kd140 μm1.5 ± 0.3 μm130 ± 5 nm18 ± 2 μm4.1 ± 0.2 μm100-200 nm2 nm/40-460 pmΔFNA72 ± 7%68 ± 12%52 ± 17%63 ± 5%NANAΔG° (kcal/mol)−5.2−7.9−9.3−6.4−7.3−9.4 to −9.0−11.7/−14.0 to −12.6ΔG°udUnitary free energies, calculated as ΔG°u = −RT ln Ka - 7.98T (33). (kcal/mol)−7.6−10.3−11.7−8.8−9.7−11.8 to −11.4−14.1/−16.4 to −15.0a From NMR titration (19Dolmer K. Huang W. Gettins P.G.W. J. Biol. Chem. 2000; 275: 3264-3271Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).b From Refs. 15Sottrup-Jensen L. Gliemann J. Van Leuven F. FEBS Lett. 1986; 205: 20-24Crossref PubMed Scopus (87) Google Scholar and 16Enghild J.J. Thøgersen I.B. Roche P.A. Pizzo S.V. Biochemistry. 1989; 28: 1406-1412Crossref PubMed Scopus (59) Google Scholar.c From single/multiple receptor model (28Arandjelovic S. Hall B.D. Gonias S.L. Arch. Biochem. Biophys. 2005; 438: 29-35Crossref PubMed Scopus (28) Google Scholar, 32Moestrup S.K. Gliemann J. J. Biol. Chem. 1991; 266: 14011-14017Abstract Full Text PDF PubMed Google Scholar).d Unitary free energies, calculated as ΔG°u = −RT ln Ka - 7.98T (33Lewis S.D. Shields P.P. Shafer J.A. J. Biol. Chem. 1985; 260: 10192-10199Abstract Full Text PDF PubMed Google Scholar). Open table in a new tab