Structural basis of BMP signaling inhibition by Noggin, a novel twelve-membered cystine knot protein

Background: The activity of bone morphogenetic proteins (BMPs) is regulated extracellularly by several families of secreted, negatively-acting factors. These BMP antagonists participate in the control of a diverse range of embryonic processes, such as establishment of the dorsal-ventral axis, neural induction, and formation of joints in the developing skeletal system. The ongoing process of neurogenesis in the adult brain also requires inhibition of BMP ligand activity. To date, the three-dimensional structures of these antagonists as well as the nature of their interaction with ligand have remained unknown. Toward that end, we have determined the crystal structure of the antagonist Noggin bound to BMP-7. Methods: The complex of the two homodimeric proteins was preformed, isolated by size exclusion chromatography, and crystallized at neutral pH. To probe the molecular interface of the complex and to quantitate the activity of a human mutant form, variant Noggin proteins were produced and their binding affinities were measured in vitro. The correlation between binding affinity and biological activity was examined with Noggin-soaked beads implanted in the developing chick limb bud. Results and Conclusions: The structure of the complex reveals that Noggin inhibits BMP signaling by blocking the binding sites of both types of receptors (Type I and Type II), mimicking their modes of binding. The affinity of Noggin variants for BMP-7 correlated well with the inhibition of BMP-induced chondrogenesis in the chick limb bud, confirming that Noggin acts by sequestering the ligand in an inactive state. Interestingly, the scaffold of Noggin was found to contain a cystine knot topology and protein fold similar to that of BMPs, indicating that ligand and antagonist may have evolved from a common ancestral gene. Clinical Relevance: Mutations in the human Noggin locus (NOG) are associated with three similar yet distinct skeletal dysplasias: proximal symphalangism (SYM1), multiple synostoses syndrome (SYNS1), and tarsal-carpal coalition syndrome (TCC). The crystal structure of the Noggin:BMP-7 complex provides a structural context for interpreting the effects of missense mutations with respect to Noggin protein folding, stability, or activity. The structure also provides the basis for engineering variants of Noggin that may have therapeutic applications in the treatment of fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder of connective tissue resulting from lymphocytic misexpression of BMPs.