Predicted structure of RBDl.

A, Comparison of the NMR-determined secondary structure for human α2M RBD (according to reference 23) with the predicted secondary structure for human α2ML1 RBDl. The heavy lines with the letter “BS” indicate regions of β sheet conformation and the dotted lines indicate regions of α-helical conformation (black color for α2M and grey color for α2ML1). The α-helix region of α2M RBD is assumed to trigger binding to LRP1. The two lysine residues (Lys1370 and Lys1374) are underlined. In α2ML1 RBDl, two distinct α-helices (denoted 1 and 2) are predicted, and the three lysine residues surrounding those regions are underlined. The S4 β-sheet is missing while the S5 β-sheet is only predicted by the method of Chou-Fasman. A hypothetical 3D model of RBDl was built using the 3D structure 1BV8 (human RBD) as template. The major helix regions are labeled in red and the β-strands are labeled in blue. Lysine residues (Lys1370 and Lys1374 for 1BV8, and Lys1392–1393 for RBDl) are labeled in green. B, α2M-MA binding at the cell surface in the presence of RBDl. RAW cells were preincubated or not (lane 1) with RBDl (lanes 2 (0.05 µM), 3 (0.1 µM), 4 (0.5 µM), 5 (0.5 µM)) or with 1 µM of GST protein (lane 6) for 30 min at 4°C before addition of α2M-MA at 0.015 µM for an additional 2 h incubation at 4°C. Immunoprecipitation and Western blot detection was performed using anti-α2M antibody. RBDl competes for α2M-MA binding in a dose-dependent manner. C, Multalin RBD alignments between representative α2M members, human α2ML1 and predicted orthologs of α2ML1. The dotted lines indicate the α-helix domains and lysine residues are marked by asterisks.