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.


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.
Groppe
Jay
J
Salk Institute, La Jolla, CA 92037, USA. groppe@salk.edu
Greenwald
Jason
J
Wiater
Ezra
E
Rodriguez-Leon
Joaquin
J
Economides
Aris N
AN
Kwiatkowski
Witek
W
Baban
Kandan
K
Affolter
Markus
M
Vale
Wylie W
WW
Izpisua Belmonte
Juan Carlos
JC
Choe
Senyon
S
D000818:Animals; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D002352:Carrier Proteins; D002454:Cell Differentiation / Q000235:genetics*; D002642:Chick Embryo; D019902:Chondrocytes / Q000166:cytology; D003460:Crystallization; D004252:DNA Mutational Analysis; D006801:Humans; D015394:Molecular Structure; D010012:Osteogenesis / Q000235:genetics*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*; D016212:Transforming Growth Factor beta; D000099176:Noggin Protein
D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D002352:Carrier Proteins; D011506:Proteins; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D000099176:Noggin Protein; C525819:BMP7 protein, human