NMR Solution Structure of Complement-like Repeat CR3 from the Low Density Lipoprotein Receptor-related Protein EVIDENCE FOR SPECIFIC BINDING TO THE RECEPTOR BINDING DOMAIN OF HUMAN a 2 -MACROGLOBULIN*

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 a 2 -macro- globulin. CR3 is one of eight related repeats that consti-tute a major ligand binding region of LRP. The structure is very similar in overall fold to homologous comple-ment-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 antipar-allel b -sheet, a one turn a -helix, and a high affinity calcium site with coordination from four carboxyls and two backbone carbonyls. The surface electrostatics and to-pography 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 ; 140 m M ), inter- action between CR3 and human a 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 circu-lation exchange chromatography to remove any residual traces of papain. Con- centrations of RBD were determined using an extinction coefficient at 280 nm of 7800 M 2 1 cm 2 1 based on the amino acid composition. NMR Spectroscopy— NMR spectra were recorded on a four channel Bruker DRX600 equipped with a pulsed-field-gradient accessory and operating at 600.13 MHz for 1 H. NMR data were processed and ana-lyzed using Triad 6.3 software (Tripos, Inc., St. Louis, MO). Experi- ments used to make resonance assignments and establish NOE connec-tivities were carried out on 2 m M 13 C/ 15 N-labeled CR3 in 20 m M d 3 - acetate, pH 5.5, 10 m M CaCl 2 , 10% D 2 O. For experiments to determine slowly exchanging amide protons, freeze-dried CR3 was reconstituted into D 2 O in the presence of 10 m M CaCl 2 and 20 m M d 3 -acetate at pH 5.5. For the titration of plasma RBD into labeled CR3, 15 N-HSQC spectra of 100 m M 13 C, 15 N-labeled CR3 were recorded in 20 m M Tris-HCl buffer, pH 7.4, containing 100 m M NaCl, 10 m M CaCl 2 , 0.1% NaN 3 after the addition of 10% D 2 O. RBD, in the identical buffer, was added to stoichiometries between 0.25 RBD:1 CR3 and 4.0 RBD:1 CR3. Standard NMR pulse sequences, as described previously for determination of the structure of the related LRP domain CR8 (16), were used for sequence assignment and determination of distance constraints. The center fre-quencies were 4.70 ppm, 118 ppm, and 43 ppm for 1 H, 15 N, and 13 C, respectively. Sensitivity enhancement gradient pulses were employed (17, 18). Structure Determination— The stucture of CR3 was determined using the same approach and experiments used for our earlier structure determination of the related LRP domain CR8 (16). In brief, sequence assignments were made by combining data from 15 N-total correlation spectroscopy-HSQC and HBHA(CO)NH