Fig. 6. ALK1 can form a complex with pro-BMP9.

a Overall structure of the human pro-BMP9:ALK1 complex at 3.3 Å (BMP9 in green, ALK1 in magenta, prodomain in orange) overlaid onto the pro-BMP9 structure (4YCG, grey, semi-transparent). b Backbones of the BMP9:ALK1 portion from the pro-BMP9:ALK1 structure (BMP9 in green, ALK1 in magenta) overlaid onto the same region in the BMP9:ALK1:ActRIIb structure (4FAO, grey). c Overlay of the two prodomains from the pro-BMP9:ALK1 structure (shown in cartoon and coloured in orange and grey respectively) and that from 4YCG (in ribbon, cyan). d In the pro-BMP9:ALK1 structure, the conserved region 2 in BMP9 makes the same four backbone H-bond interactions with the prodomain as shown in Fig. 5e. Four residues in the conserved region 2 are shown in blue spheres and labelled with BMP10 numbering. e–h Analysis of complex formation by analytical gel filtration. Purified pro-BMP9, pro-BMP9 mixed with ALK1, pro-BMP9 mixed with sENG and sENG were run separately on an S200 10/300 gel filtration column which was pre-equilibrated with 20 mM Tris.HCl, 150 mM NaCl, pH 7.4. e Gel filtration traces. The arrows indicate the elution volumes of the standards. Numbers 1-6 indicate the 6 peaks which were analysed by SDS-PAGE. f Middle fraction from each peak was run on an SDS-PAGE. Identities of the proteins on the SDS-PAGE are indicated using coloured circles. g Consecutive fractions from each gel filtration experiment were run on a non-reducing SDS-PAGE and immunoblotted using an anti-BMP9 prodomain antibody. Each analytical sample run was repeated at least one more time with fraction checked on SDS-PAGE to ensure reproducibility. h Cartoon diagrams, using the same colouring scheme as the circles in f, to illustrate that mixing pro-BMP9 with ALK1 leads to the formation of pro-BMP9:ALK1 complex, whereas mixing pro-BMP9 with sENG leads to the displacement of the prodomain which can be readily detected as a different peak in the gel filtration.