Transduction of temporally and spatially specific signals through the BMP pathway in a diverse array of cellular contexts is achieved in part by differential affinities of the receptors of the heterotetrameric-signaling complex for the array of ligands in the family. However, differential affinity to secreted inhibitors that act as ligand traps plays a reciprocal, equally important role in context-specific tuning of BMP signals. Here, we show that single point mutations (N445K,T) in the wrist epitope of the BMP-related ligand GDF5 lead to a resistance to Nog, and a subsequent dramatic increase in the intensity of the signaling output. Moreover, we show that wildtype BMP9 and BMP10, two divergent members of the family that harbor the asparagine for lysine substitution, are markedly resistant to Nog in limb bud micromass and C2C12 myoblast cell assays.
The GDF5 mutations N445K,T are associated with an inherited skeletal disease characterized by joint fusions in fingers, toes and elbows. This phenotype is similar to that of SYNS1, previously associated with hypomorphic alleles of NOG
. SYM1, a less severe condition in which symphalangism is limited to the small interphalangeal joints of the fourth and fifth finger, is also caused by mutations in NOG
. We have previously shown that a SYM1-like phenotype can also result from gain of function mutations in GDF5 
GDF5 mutations described here affect the wrist epitope, which is the polypeptide segment between the third and fourth conserved cysteine residue that is most divergent as shown in the multiple sequence alignment of TGFβ superfamily ligands (). Because the wrist epitope is a key element of the type I receptor interface, the divergence may reflect the differential affinity of the superfamily of signaling ligands for their specific receptors. In addition, the diversity may reflect differential affinity for the array of secreted antagonists. For example, TGFβ ligands are deposited extracellularly as an inactive latent complex not regulated by secreted antagonists, whereas BMP/GDF ligands can bind with picomolar affinity, or evade inhibition almost entirely, as we have shown here for the interaction of NOG with the divergent BMP9, BMP10 pair 
Functional studies of the N445K,T mutations suggest a dual effect of the mutations - an altered signaling effect on the one hand and resistance to NOG on the other. Whereas the insensitivity of N445K,T mutations was clearly demonstrated in retroviral overexpression studies or by stimulation with recombinant proteins, the altered signaling effect could not be finally clarified on a molecular level. The fact that - in contrast to rhGDF5 - the rhN445T GDF5 mutant induced significant amounts of ALP in C2C12 cells prompted us to speculate on altered receptor specificity. This hypothesis was further analyzed by neutralization assays of different TGFbeta type 1 receptors and Biacore analyses. RhN445T GDF5 seemed to be slightly more sensitive to the inhibition by BMPR1Aecd and BMPR1Becd, but no significant changes of receptor affinities were detected. The different signaling effect might be explained either by different association and dissociation rates of the ligand-receptor complex or by the involvement of other co-receptors.
Interestingly, the reported R438L mutation in GDF5 lies within the same domain, i.e. the wrist epitope (type I receptor interface as well as NOG binding interface). Yet it results in altered receptor specificity with increased affinity to the BMPR1A, and not affecting the inhibition by NOG. The R438 position is not conserved within the BMP/GDF subgroup. We demonstrated that the rhR438L GDF5 mutant has a higher affinity for the BMPR1A receptor than rhGDF5, giving this mutant BMP2-like properties. BMP2 is also inhibited by NOG, which indicates that this amino acid change seems to have an effect on the receptor specificity only, but otherwise it is well tolerated by the BMP antagonist NOG.
The similarity of the resulting phenotypes caused by either loss of function NOG mutations or activating mutations in GDF5 argues in favour of GDF5 representing the most important target for NOG during the critical phase of joint development and indicates that SYM1/SYNS1 are associated with an increased biological activity of GDF5.
The crystal structure of the NOG-BMP7 complex showed that NOG, a covalently linked homodimer, inhibits BMP signaling by blocking the molecular interfaces of the binding epitopes for both type I and type II receptors, sequestering the covalently linked homodimeric ligand in an inactive complex 
. In the crystal structure of GDF5 
, the conserved asparagine (N445) is located near the amino terminal end of the large helix (α3), its amide sidechain projecting tangential to, and hydrogen bonding with, the backbone of the long finger 2 at a main chain carbonyl (E491) across the dimer interface. Superposition of the GDF5 crystal structure on BMP7 in the NOG-BMP7 complex provides a model of the NOG-GDF5 complex for interpretation of the role of the conserved asparagine and the consequences of the mutations. In the model, just as in the NOG-BMP7 crystal structure, the amide nitrogen of the asparagine sidechain is hydrogen bonded to two main chain carbonyl groups, one across the dimer interface (GDF5 E491) and one along the extended N-terminal 14 clip of the antagonist (NOG A36). In addition to this triangulated interaction, the carbonyl oxygen of the N445 sidechain also forms hydrogen bonds with the main chain via the amide nitrogen of NOG A36. Furthermore, in a superposition model of a GDF5-type I receptor complex, the asparagine sidechain appears to interact with E81 and G82 across the ligand-receptor interface. Thus, loss of the amide group by substitution with a lysine or threonine side chain in the N445K,T mutants would on the one hand disrupt two stabilizing interactions within the NOG-GDF5 complex, and, on the other hand, alter the ligand-receptor interface resulting in a broader specificity. With respect to the differential effects of threonine substitution on interaction with NOG, superposition of GDF5 and BMP7 in the crystal structure of the BMP7-NOG complex reveals a major structural difference between the two ligands at the interface with the key residue of the N-terminal extension of NOG, P35, also a site of several human mutations linked to SYM1 
. Due largely to major conformational differences in the backbone and side chains of the short finger 1 of GDF5 relative to the BMPs, the hydrophobic pocket that accommodates the proline side chain is predicted to be only half formed, which would disrupt the stabilizing interaction to a similar extent as mutation of P35. Thus, interaction between wildtype GDF5 and NOG is anticipated to be weaker than that between BMPs and NOG, as evidenced by our titration assays which required super-stoichiometric ratios (cf.
), perhaps so much so that disruption of the adjacent interaction between the conserved asparagine and the backbone of the N-terminal extension (A36) is sufficient to abolish formation of the GDF5-NOG complex. BMP9 and BMP10, which are not responsive to inhibition by NOG, do not bear the homologous asparagine residue. However introduction of the asparagine in BMP9 was not sufficient to confer sensitivity to NOG. Successive introduction of two additional GDF5-like substitutions, removal of a BMP9-specific insert (YH415Q) and K348L, imparted near total sensitivity. In conclusion, while substitution of the single asparagine is sufficient to impart NOG sensitivity in GDF5, other BMPs require further structural alteration in order to impart sensitivity, or resistance. Because ligand-receptor and ligand-antagonist complexes are sufficiently structurally conserved within the BMP/GDF family, GDF5 function was successfully transferred to BMP9 by rounds of rational design.
Currently, rhBMP2, rhBMP7, and rhGDF5 are used in clinical applications for bone regeneration or are under investigation in clinical trials. Although BMPs are very potent in cell culture systems or small animal models (nanograms), significantly higher concentrations are needed for measurable effects in humans (milligrams). It was shown that BMP antagonists are regulated during fracture healing 
, indicating that endogenous regulation of the BMP signaling cascade may neutralize exogenously applied rhBMPs, necessitating higher doses than required in vitro. In keeping with this hypothesis, the in vivo and in vitro studies presented here demonstrated induction of Nog after treatment with rhBMPs, an apparent physiological response to regulate and constrict BMP action. The upregulation of inhibitors in response to exogenous growth factors may therefore also be applied for BMP treatment, which can only be partially compensated for by high dosage due to feedback control mechanisms 
. Thus, variant BMPs that are insensitive to antagonist may induce bone formation more effectively, providing a source for effective, low-dose therapeutics for clinical applications.