Fig. 4: hTAAB IgG binding mediates polygonal assembly of Tie2 dimers.

a Immunoblot analysis for relative phosphorylation ratios of Tie2 after treatment of HUVECs with chimeric hTAAB Fab or hTAAB mouse IgG1 (1 and 10 μg/ml) (left). Densitometric analyses of p-Tie2/Tie2 ratios are shown (right). Data from three independent experiments (n = 3) were analyzed and expressed as mean ± SD (***P < 0.001 vs. control). b Immunoblot analysis for relative phosphorylation ratios of Akt after treatment of HUVECs with chimeric hTAAB Fab or hTAAB IgG1 (1 and 10 μg/ml) (left). Densitometric analyses of the p-Akt/Akt ratios are shown (right). Data from three independent experiments (n = 3) were analyzed and expressed as mean ± SD (*P = 0.0304, ***P < 0.001 vs. control). a, b P values by one-way ANOVA test followed by Dunnett’s multiple comparisons test. ns, not significant. c Size-exclusion chromatography-multiangle light-scattering (SEC-MALS) analysis of hTie2 Fn2–3, hTie2 ECD, hTAAB/hTie2 Fn2–3 complex, and hTAAB/hTie2 ECD complex. Samples were run on a Superdex 200 Increase 10/300 GL column in PBS at 0.5 ml/min, at protein concentrations of 3 mg/ml (hTie2 Fn2–3) or 1 mg/ml (all others). The molecular mass (kDa) and absorbance at 280 nm are plotted against elution volume (ml). d Design of the Tie2 dimeric mutant. Residues D682 and N691 (yellow) on both ends of the antiparallel β-sheet interaction interface between Fn3 domains were mutated to cysteine to allow the formation of two disulfide bonds, thus generating a constitutive Tie2 Ig3–Fn3 dimer. e Purified hTie2 Ig3–Fn3 D682C/N691C was analyzed by SDS-PAGE and Coomassie blue staining under reducing and non-reducing conditions. Similar results were observed in three independent experiments. f Size-exclusion chromatography of hTie2 Ig3–Fn3 D682C/N691C in complex with hTAAB IgG1. Purified hTie2 Ig3–Fn3 D682C/N691C was incubated with hTAAB IgG1 for 2 h at a molar ratio of 2:1 (Tie2 monomer:hTAAB IgG1) and applied to size-exclusion chromatography. The fraction used for negative-stain EM and 2D class average is indicated as EM analysis. g Representative 2D class averages of Tie2 dimeric mutant/hTAAB IgG1 complex (Top). Cyclical higher-order structure of the Tie2 dimer/hTAAB IgG1 complex in 4-to-4, 5-to-5, and 6-to-6 assemblies were modeled based on the 2D class averages of tetragonal, pentagonal, and hexagonal closed-ring structures, respectively, using crystal structures of the Tie2 Fn2–3/chimeric hTAAB Fab complex, Tie2 Ig1–Fn1 (PDB: 4K0V¹¹) and Fc fragment of human IgG1 (PDB: 5VGP⁵⁷) (bottom). Scale bars, 20 nm. h 3D Model of the polygonal 5-to-5 assembly of the Tie2 dimer/hTAAB IgG1 complex observed on the negative-stain EM grid. The 2:2 Tie2 Fn2–3/chimeric hTAAB Fab complex in an asymmetric unit of the crystal is indicated by a red line.