FIGURE 5.

Identification of a FLNa-binding motif within the N-terminal peptide of ASB2α. A, combined sequence and secondary structure alignment of ASB2α peptide (residues 1–20) against integrin β2 (residues 747–765), integrin β7 (residues 770–789), migfilin (residues 1–20) peptides, and their respective IgFLNa21-binding motifs solved by crystallography (PDB codes 2JF1_T, 2BRQ_C, and 2W0P_C). Predicted and x-ray-derived (indicated by arrows) locations of β-strands were in good agreement, validating the method of the secondary structure prediction used by Align 123. B and C, main structural determinants of the interaction between IgFLNa21 and either the integrin β7 (B) or migfilin (C) motifs, as derived from crystallographic structures (respective PDB codes 2BRQ and 2W0P). Interacting residues color code: red for IgFLNa21 (Leu-2271, Ile-2273, Val-2275, Ile-2283, and Phe-2285), brown for integrin (Tyr-778, Ile-782, and Thr-784) or migfilin (Val-9, Val-13, and Ile-15), and pink for Ser-780 or Ser-11. D, modeled structure resulting from docking ASB2α N-terminal peptide into the IgFLNa21 CD groove. The display code used was described for B and C. E, ASB2αANK(1–10) associates with IgFLNa21. Protein extracts from HeLa cells mock transfected (lane 1) or transfected with the GFP-ASB2αANK(1–10) vector (lanes 2–5) were immunoprecipitated (IP) with anti-ASB2 serum (lanes 1, 2, 4, and 5) or with control ASB2 preimmune serum (lane 3). Immobilized proteins were further incubated with extracts from E. coli expressing GST-IgFLNa21 (lanes 1–4) or GST alone (lane 5). The interacting complexes, as well as aliquots of E. coli extracts (E. coli ext.), were resolved by SDS-PAGE and immunoblotted with anti-GST and/or anti-ASB2 antibodies. The heavy (IgH) and light (IgL) chains of immunoglobulins are indicated. F, ASB2αANK(1–10) binds IgFLNa directly. ASB2αANK(1–10)-His₆ and GST-IgFLN proteins were purified by affinity chromatography. 0.2 μg of ASB2αANK(1–10)-His₆ (lane 1), 5 μg of GST-IgFLNa21 (lane 2), 5 μg of GST-IgFLNa21AA/DK (lane 3), and 5 μg of GST-IgFLNb21 (lane 4) were run on a SDS-PAGE. The gel was stained with Coomassie Blue. Direct pulldown assays were performed using purified ASB2αANK(1–10)-His₆ and glutathione beads that were uncoated (lane 5) or coated with purified GST-IgFLNa21 (lane 6), GST-IgFLNa21AA/DK (lane 7), and GST-IgFLNb21 (lane 8). Bound proteins were detected by Ponceau staining (GST fusion proteins) and immunoblotting with anti-ASB2 antibodies (ASB2αANK(1–10)-His₆). Lane 9, molecular mass marker.