FIGURE 5.

Site III interactions at the ALK1/BMP9 interface. A, close-up of the interaction surface, with color scheme retained from Fig. 3. ALK1 specificity residues His⁷³ and Glu⁷⁵ and Lys⁵³ from BMP9 are highlighted, with polar contacts drawn as dashed lines. B, overlay of the α1 helix from ALK1 and ALK3 showing the positioning of Glu⁷⁵ (ALK1) with respect to Asp⁸⁴ (ALK3) and Phe⁸⁵ (ALK3). C, sequence alignment of type I receptors (ALK1–7), with ALK1 residues His⁷³ and Glu⁷⁵ highlighted in gold. D, sequence alignment of BMP9 and BMP10 with select TGF-β ligands, showing the invariant lysine residue Lys⁵³ (blue). Asterisks coincide with panel A. E, cell based, and F, SPR analysis of ALK1 mutants showing effects of His⁷³ and Glu⁷⁵ substitutions on BMP9 binding and signaling. Proteins were expressed in COS-1 cells, purified by affinity chromatography and showed similar purity (SDS-PAGE given in the upper right panels shows: MW markers (first lane), ALK1-WT (second lane), ALK1(E75F) (third lane), ALK1(E75V) (fourth lane), and ALK1(E75A (fifth lane). SPR analysis was performed with Biacore T100 at 37 °C. Proteins were captured on ani-hFC IgG chip at similar levels. Positive control (WT ALK1) and negative control (unrelated protein that does no bind to BMP9) are given for comparison.