Structure of the ternary signaling complex of a TGF-beta superfamily member

Abstract

The crystal structure of the complete signaling complex formed between bone morphogenetic protein 2 (BMP-2) and the extracellular domains (ECDs) of its type I receptor [bone morphogenetic protein receptor type Ia (BMPR-Ia)-ECD] and its type II receptor [activin receptor type II (ActRII)-ECD] shows two fundamental structural constraints for receptor assembly. First, the homodimeric BMP-2 ligand assembles two pairs of each receptor symmetrically, where each of the receptor ECDs does not make physical contact. Therefore, conformational communication between receptor ECDs, if any, should be propagated through the central ligand. Second, the type I and II receptor interfaces of the complex, when compared with those of binary complexes such as BMP-2/BMPR Ia-ECD, BMP-7/ActRII-ECD, and activin/ActRIIb-ECD, respectively, show there are common sets of positions repeatedly used by both ligands and receptors. Therefore, specificity-determining amino acid differences at the receptor interfaces should also account for the disparity in affinity of individual receptors for different ligand subunits. We find that a specific mutation to BMP-2 increases its affinity to ActRII-ECD by 5-fold. These results together establish that the specific signaling output is largely determined by two variables, the ligand-receptor pair identity and the mode of cooperative assembly of relevant receptors governed by the ligand flexibility in a membrane-restricted manner.

Fig. 1.

Ternary complex of BMP-2/BMPR-Ia-ECD/ActRII-ECD shows noncontacting assembly of receptors. (a) Ternary complex of BMP-2/BMPR-Ia-ECD/ActRII-ECD as seen in the membrane. BMP-2 dimer subunits are shown in red and orange, displaying the butterfly conformation. The two BMPR-Ia-ECDs are shown in blue, and the ActRII-ECDs are shown in green. Dashed lines represent C-terminal residues connecting to the membrane-spanning segments of the receptors. Cystines are shown as yellow spheres. (b) A top view of the complex. The color scheme is the same as in a. Labeled are α-helix 3 of BMP-2 and α-helix 1 of BMPR-Ia. (Inset) The M loop of the ActRII from BMP-2/ActRII-ECD/BMPR-Ia-ECD (green), BMP-7/ActRII-ECD (blue), and activin/ActRIIb-ECD (red) structures.

Fig. 2.

Ligand-binding interfaces of type II receptors show a nearly common set of interface positions. Peeled-away interfaces of BMP-2/ActRII-ECD (a), BMP-7/ActRII-ECD (b), and activin/ActRIIb-ECD (c) pairs shown as space-filling model. Orientation is as in Fig. 1a. Colored are those in contact between the two chains, for identical (pink), highly conserved (green), and nonconserved (blue) residues. Asterisks denote reference contact points between the two molecules. (d) List of the five identical residues in ActRII and ActRIIb and their contacting amino acids on three ligands.

Fig. 3.

Ligand-binding interfaces of type I receptors are formed at the junction between two ligand monomers. (a) Peeled-away interfaces of BMP-2/BMPR-Ia-ECD of the ternary complex. Residues from two BMP-2 subunits (red and orange) and those from BMPR-Ia-ECD (blue) are shown in the standard orientation (Fig. 1a). (b) Peeled-away interfaces of BMP-2/BMPR-Ia-ECD of the binary complex (20). Only those amino acids that were discussed for two pockets are labeled. All other residues are listed below.

Fig. 4.

Receptor-binding specificity can be altered by local changes at its interface. (a) Biacore affinity data of BMP-2 mutants to ActRII-ECD. Affinities are shown in nanomolar concentrations calculated as k_off/k_on. (b) Closeups of the three BMP-2 mutants showing increased affinity for ActRII-ECD. Residues are shown in ball-and-stick representation for BMP-2 (orange) and ActRII-ECD (green). Shown in red is the modeled conformation of the mutant sidechains of BMP2_S85R (1), BMP2_L100K (2), and BMP2_E109R (3). In box 2, the M loop based on BMP-7/ActRII-ECD structure is shown in gray. H bonds and charge pairs are shown as dashes (nitrogen, blue; oxygen, red; and sulfur, yellow).