Specificity and structure of a high affinity activin receptor-like kinase 1 (ALK1) signaling complex

Abstract

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.

FIGURE 1.

Kinetic and thermodynamic analysis of BMP9 binding to ALK1, ActRIIB, ActRIIA, and BMPRII. A, SPR sensograms of ALK1^ECD-Fc, ActRIIB^ECD-Fc, ActRIIA^ECD-Fc, and BMPRII^ECD-Fc binding to BMP9 at 25 and 37 °C. Raw data (colored lines) were overlaid with a global fit to a 1:1 model with mass transport limitations (black lines). ActRIIA^ECD-Fc data were analyzed using affinity fit in SigmaPlot. B, kinetic parameters for receptor-ligand interactions. C, comparison of thermodynamic parameters of ALK1^ECD-Fc, ActRIIB^ECD-Fc, and ActRIIA^ECD-Fc interactions with BMP9 at 25 °C.

FIGURE 2.

Kinetic and thermodynamic analysis of BMP10 binding to ALK1, ActRIIB, ActRIIA, and BMPRII. A, SPR sensograms of ALK1^ECD-Fc, ActRIIB^ECD-Fc, ActRIIA^ECD-Fc, and BMPRII^ECD-Fc binding to BMP10 at 25 and 37 °C. B, kinetic parameters for receptor-ligand interactions. C, comparison of thermodynamic parameters of ALK1^ECD-Fc, ActRIIB^ECD-Fc, and ActRIIA^ECD-Fc interactions with BMP10 at 25 °C.

FIGURE 3.

Structure of the ALK1^ECD-BMP9-ActRIIB^ECD ternary complex. A, the ternary complex comprised of one BMP9 homodimer (red/blue), two ALK1^ECD receptors (gold), and two ActRIIB^ECD receptors (green). The complex is shown as viewed from above (left) and from the side with the membrane below (right). B, superposition with the BMP2-ALK3^ECD-ActRIIB^ECD ternary complex (shown in ribbon) via the ligands reveals differences in receptor positioning (BMP2 homodimer colored pink/light blue; ALK3^ECD and ActRIIB^ECD receptors colored light gray and dark gray, respectively; ALK1^ECD-BMP9-ActRIIB^ECD complex colored as in panel A). C, superposition of ALK1^ECD and ALK3^ECD at the BMP9 interface (shown in surface representation) reveals a 20° rotation of the ALK1 protomer pivoted around helix α1. D, close-up view of helix α1, F1, F2, and F3 loops from ALK^ECD and ALK3^ECD (shown as schematic). Repositioning of these elements in ALK1 results in a novel set of receptor-ligand interactions.

FIGURE 4.

Site I and II interactions at the ALK1/BMP9 interface. A, close-up of the interaction surfaces, with the color scheme retained from Fig. 3. ALK1 is shown in schematic, with key residues drawn as sticks; BMP9 is shown in surface representation. For clarity the overall complex is shown to the side. B, detailed close-up of the interfaces, showing specific polar contacts (dashed lines) between residues along the F2 and F3 loops of ALK1. Key residues are highlighted with asterisks. C, sequence alignment of type I receptors (ALK1–7), with contact residues highlighted in gold. D, sequence alignment of BMP9 and BMP10 with select TGF-β ligands, showing key contact residues from both BMP9 protomers (red and blue). Asterisks coincide with panel B.

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.

FIGURE 6.

ActRIIB/BMP9 interface and basis for ActRIIB selectivity. A, peeled-away surface of ActRIIB showing residues involved in BMP9 binding. Polar (blue) and hydrophobic (gray) contacts are highlighted. B, structure-based sequence alignment of ActRIIB with ActRIIA. ActRIIB contacts are colored as in panel A. C, insertion of Tyr⁹⁷ in BMP9 causes a loop out in the backbone structure. D, interaction of ActRIIB residues Arg¹⁴, Lys³⁰, and Ser⁸⁰ with BMP9 (residues shown at sticks). Alignment of the corresponding residues from ActRIIA (colored gray) suggests a mechanism for receptor discrimination. Asterisks coincide with panel B.

FIGURE 7.

Mapping HHT2 mutations to the ALK1 structure. A, surface mapping of HHT2 mutations (Cys residues shown in blue; non-Cys residues in gray; BMP9 contact residues in red). A corresponding list of HHT2 mutations is shown to the right. B, close-up view of stabilizing interactions for ALK1 residues Gly⁴⁸, Ala⁴⁹, Trp⁵⁰, and Thr⁵² and C, Arg⁶⁷ and Asn⁹⁶.