Crystal Structure of BMP-9 and Functional Interactions with Pro-region and Receptors

Abstract

Bone morphogenetic proteins (BMPs), a subset of the transforming growth factor (TGF)-␤ superfamily, regulate a diverse array of cellular functions during development and in the adult. BMP-9 (also known as growth and differentiation factor (GDF)-2) potently induces osteogenesis and chondrogenesis, has been implicated in the differentiation of cholinergic neurons, and may help regulate glucose metabolism. We have determined the structure of BMP-9 to 2.3 Å and examined the differences between our model and existing crystal structures of other BMPs, both in isolation and in complex with their receptors. TGF-␤ ligands are translated as precursors, with pro-regions that generally dissociate after cleavage from the ligand, but in some cases (including GDF-8 and TGF-␤1, -2, and -3), the pro-region remains associated after secretion from the cell and inhibits binding of the ligand to its receptor. Although the proregion of BMP-9 remains tightly associated after secretion, we find, in several cell-based assays, that the activities of BMP-9 and BMP-9⅐pro-region complex were equivalent. Activin receptor-like kinase 1 (ALK-1), an orphan receptor in the TGF-␤ family, was also identified as a potential receptor for BMP-9 based on surface plasmon resonance studies (BIAcore) and the ability of soluble ALK-1 to block the activity of BMP-9⅐pro-region complex in cell-based assays.Transforming growth factor ␤ (TGF-␤) 1 signaling controls a wide variety of processes over the lifetime of an organism. A subset of this large and well conserved family is the group of bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs), which regulate a diverse array of cellular functions, including differentiation, proliferation, organogenesis, axon guidance, apoptosis, and the establishment of left-right asymmetry (1-3). BMPs and GDFs are highly conserved throughout the animal kingdom, with examples ranging from Drosophila to humans. They have frequently been implicated in the treatment of bone disorders and injury, in accordance with their robust ability to generate de novo bone formation.All TGF-␤ ligands are translated as precursor proteins, consisting of an amino-terminal pro-region and a carboxyl-terminal ligand. This precursor forms a disulfide-linked homodimer in the cytoplasm, and the pro-region is then cleaved from the ligand. In most cases, the pro-region disassociates, and the mature ligand is secreted from the cell, but the pro-regions of GDF-8 (also known as myostatin) and TGF-␤1, -2, and -3 remain noncovalently associated with the ligand after secretion and inhibit binding of their ligands to their respective receptors (4 -6). Transgenic mice overexpressing the pro-region of GDF-8 show dramatic increases in muscle mass, further indicating that the pro-region functionally inhibits GDF-8 (7). The proregion of BMP-9 also remains tightly associated after secretion from the cell.BMP signaling is induced when a dimeric ligand binds to the extracellular domains of two type I and two type II receptors (8). This assembl...

Structure of bone morphogenetic protein 9 procomplex

“…4F). The EC 50 values for type I and II receptors agree with previous affinity measurements with BMP9 (16,17). Interestingly, the BMP7 prodomain also competes binding of BMP7 to type II but not type I receptors (18), consistent with the open-armed conformation of pro-BMP7 (Fig.…”

Anti-human Activin Receptor-like Kinase 1 (ALK1) Antibody Attenuates Bone Morphogenetic Protein 9 (BMP9)-induced ALK1 Signaling and Interferes with Endothelial Cell Sprouting

“…Furthermore, in vivo angiogenesis was promoted by BMP9 in a Matrigel plug assay and in a BxPC3 xenograft model of human pancreatic cancer (34). A possible explanation lies in the fact that BMP9 may bind other receptors at higher concentrations that could have opposing effects on angiogenesis (37). Although the concentration of circulating BMP9 was reported to vary between 2 and 12 ng/ml in sera and plasma from healthy humans (33), the concentration of BMP9 in the proangiogenic tumor microenvironment is currently not known.…”

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

“…wrist-helix, although we see that the wrist-helix from chain 2 for each dimer docks in approximately the same location onto chain one. This repositioning of the structure alters the conformation from the butterfly like dimer reported previously for myostatin 44 and other TGF-b family proteins 49 ( Fig. 3d) to a more linear dimer (Fig.…”