The low density lipoprotein receptor-related protein is a member of the low density lipoprotein receptor family and contains clusters of cysteine-rich complementlike repeats of about 42 residues that are present in all members of this family of receptors. These clusters are thought to be the principal binding sites for protein ligands. We have expressed one complement-like repeat, CR8, from the cluster in lipoprotein receptor-related protein that binds certain proteinase inhibitor-proteinase complexes and used three-dimensional NMR on the 13 C/ 15 N-labeled protein to determine the structure in solution of the calcium-bound form. The structure is very similar in overall fold to repeat 5 from the low density lipoprotein receptor (LB5), with backbone root mean square deviation of 1.5 Å. The calcium-binding site also appears to be homologous, with four carboxyl and two backbone carbonyl ligands. However, differences in primary structure are such that equivalent surfaces that might represent the binding interfaces are very different from one another, indicating that different domains will have very different ligand specificities.The low density lipoprotein receptor-related protein (LRP) 1 is a member of the low density lipoprotein receptor family (1). Members of this family of receptors contain a small cytoplasmic domain, a single membrane-spanning helix, and clusters of two types of small cysteine-rich repeats on the extracellular side, interspersed with regions that contain a YWTD motif. The two types of cysteine-rich repeat are an epidermal growth factorlike repeat of about 50 residues and a complement-like repeat, so named for its presence in complement components C8 and C9, of about 42 residues. Each of these types of repeat contains 6 cysteines that are involved in three intradomain disulfide bonds. The limited evidence on the location of protein ligandbinding sites indicates that such sites are restricted largely to the clusters of complement-like repeats. The LDL receptor contains one cluster of seven such repeats and binds various apolipoproteins, including apoE and apoB100 (2). LRP contains a total of 31 such repeats organized into four clusters of 2, 8, 10, and 11 repeats (3). These clusters have been designated clusters I, II, III, and IV, respectively. LRP binds a much wider range of protein ligands including apolipoproteins, serpin-proteinase complexes, and ␣ 2 -macroglobulin-proteinase complexes (1). Studies on truncated LRP species suggest that cluster II is a major locus for protein ligand binding (4).Although structures of three complement-like repeats have been reported from the LDL receptor, one by x-ray crystallography (5) and two by NMR spectroscopy (6, 7), no structure of such a repeat has previously been reported from LRP. Both because of the sequence differences between repeats in regions that might form the ligand-binding sites and also because of the more extensive range of protein ligands that LRP can bind compared with the LDL receptor, it is important to make structural comparisons betw...

We have determined the X-ray crystal structure to 1.8 A resolution of the Ca(2+) complex of complement-like repeat 7 (CR7) from the low-density lipoprotein receptor-related protein (LRP) and characterized its calcium binding properties at pH 7.4 and 5. CR7 occurs in a region of the LRP that binds to the receptor-associated protein, RAP, and other protein ligands in a Ca(2+)-dependent manner. The calcium coordination is identical to that found in LB5 and consists of carboxyls from three conserved aspartates and one conserved glutamate, and the backbone carbonyls of a tryptophan and another aspartate. The overall fold of CR7 is similar to those of CR3 and CR8 from the LRP and LB5 from the LDL receptor, though the low degree of sequence homology of residues not involved in calcium coordination or in disulfide formation results in a distinct pattern of surface residues for each domain, including CR7. The thermodynamic parameters for Ca(2+) binding at both extracellular and endosomal pHs were determined by isothermal titration calorimetry for CR7 and for related complement-like repeats CR3, CR8, and LB5. Although the drop in pH resulted in a reduction in calcium affinity in each case, the changes were very variable in magnitude, being as low as a 2-fold reduction for CR3. This suggests that a pH-dependent change in calcium affinity alone cannot be responsible for the release of bound protein ligands from the LRP at the pH prevailing in the endosome, which in turn requires one or more other pH-dependent effects for regulating protein ligand release.

We have used NMR methods to determine the structure of the calcium complex of complement-like repeat 3 (CR3) from the low density lipoprotein receptor-related protein (LRP) and to examine its specific interaction with the receptor binding domain of human ␣ 2 -macroglobulin. CR3 is one of eight related repeats that constitute a major ligand binding region of LRP. The structure is very similar in overall fold to homologous complement-like repeat CR8 from LRP and complement-like repeats LB1, LB2, and LB5 from the low density lipoprotein receptor and contains a short two-strand antiparallel ␤-sheet, a one turn ␣-helix, and a high affinity calcium site with coordination from four carboxyls and two backbone carbonyls. The surface electrostatics and topography are, however, quite distinct from each of these other repeats. Two-dimensional 1 H, 15 N-heteronuclear single quantum coherence spectra provide evidence for a specific, though relatively weak (K d ϳ 140 M), interaction between CR3 and human ␣2-macroglobulin receptor binding domain that involves a contiguous patch of surface residues in the central region of CR3. This specific interaction is consistent with a mode of LRP binding to ligands that uses contributions from more than one domain to generate a wide array of different binding sites, each with overall high affinity.The low density lipoprotein receptor-related protein (LRP) 1 is a member of the low density lipoprotein receptor (LDLR) family and is responsible for binding and clearing from circulation a wide range of diverse protein ligands. These ligands include proteinases complexed with ␣ 2 -macroglobulin (␣ 2 M) (1, 2) and with various serpins, such as antithrombin, ␣ 1 -proteinase inhibitor, and C1 inhibitor (3, 4), various lipoproteins and lipoprotein-containing particles (5, 6), and an array of unrelated proteins such as Pseudomonas exotoxin, lactoferrin, and ␤-amyloid precursor protein. It is therefore not surprising that LRP is critical for life (7) and may be involved in the normal regulation of both proteinase and lipid metabolism.All members of the LDLR family are mosaic proteins composed of the same building blocks and with similar topological organizations (8). These components are two classes of cysteine-rich small domains (epidermal growth factor-like repeats and complement-like repeats (abbreviated here as CR)), clusters of six domains containing YWTD motifs (9), a single transmembrane helix, and a cytoplasmic domain that contains one or two copies of the NPXY motif, which is a common internalization signal. The major structural differences between family members are in the size of the protein and therefore the number of constituent domains. LRP is the largest member of the family identified so far, with molecular mass of ϳ600 kDa, compared with a molecular mass of only 180 kDa for LDLR itself. It contains 31 CR domains, which occur in four clusters of 2, 8, 10, and 11 repeats, counted from the N terminus. As with other LDLR family members, it is believed that protein ligands bind primarily t...

The American horseshoe crab Limulus polyphemus contains a,-macroglobulin (a,M) in the hemolymph plasma and hemocytes. a,M from Lirnulus shows many of the typical characteristics of mammalian a,M, including the presence of an internal thiol-ester, reactivity with a diversity of endopeptidases, a unique proteinase-trapping mechanism, and reactivity with the mammalian a,M receptor. Additionally, Limulus rx, M has the unique property that it regulates the limulin-based hemolytic system of the plasma.A cDNA encoding Limulus a,M has been obtained from a hemocyte cDNA library. The open reading frame encodes an N-terminal signal sequence of 25 amino acid residues and a mature protein of 1482 residues. The entire amino acid sequence is similar to those of the mammalian uZMs (28-29% identity) and contains common features found in mammalian a,Ms, a bait region, an internal thiol-ester site, and a receptor-binding domain. However, the N-terminal portion (positions 24-105) has no sequence similarity with those of mammalian a,Ms, and it is structurally related to that of the human complement factor C8y chain, consistent with a role for Limulus a,M in host defense. The component sugar analysis of Limulus a,M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian u,M. However, unlike mammalian m2M, no sialic acid was detected in Limulus rx,M and it contained approximately 3 mol/mol N-acetylgalactosamine, suggesting the presence of 0-linked sugar chains, which have not been found in mammalian n,M.Expression of a,M was detected in hemocyies, but not in hepatopancreas, heart, stomach, intestine, coxal gland, brain and skeletal muscle. Furthermore, immunoblotting of large and small granules of the hemocytes with antiserum against a,M indicated the presence of the cx,M in large granules. Trypsintreated Linzu/us a2M, but not the native a2M, displaced methylamine-treated human ' "51-u2M from the human cx,M receptor with a Kd of 30 nM, suggesting conservation of the proteinase-clearance mechanisms between mammalian and arthropod evolutionary lineages.Keyuvrds: u2-macroglobulin; horseshoe crab; invertebrate; cDNA.Proteins of the az-macroglobulin (a,M) family are abundant components in the plasma of mammals (Sottrup-Jensen, 1987, 1989 and arthropods (Armstrong et al., 1996;, comprising about 3% of the total plasma protein of humans (Ganrot and Schersten, 1967) and the third most abundant protein in the plasma of the American horseshoe crab, Linzulzcs polyphemus Ci,r.r.f,sl,o~zde,zc.e