Understanding of the different roles of Noggin in the Noggin-BMP-2 and Noggin-BMP-9 dimer complexes at the molecular level.

Bone morphogenetic proteins (BMPs) play crucial roles in tissue development and repair as growth factors. However, the clinical application of some type of BMPs is restricted due to their undefined and uncontrolled complex signaling pathways. This study investigates the interactions of BMPs, particularly BMP-2 and BMP-9, with Noggin, a BMP antagonist crucial for embryonic development. BMPs signal through heterohexameric receptor complexes, activating Smad and non-Smad pathways. Noggin, with its cysteine-knot structure, inhibits BMP signaling by preventing receptor binding. Molecular dynamics simulation studies of structurally similar BMP proteins show higher fluctuations in the Noggin/BMP-9 complex than the Noggin/BMP-2 complex. Surface Plasmon Resonance (SPR) analysis revealed that the equilibrium dissociation constant (K(D)) for BMP-2 indicated a 1000-fold higher binding affinity compared to BMP-9. Comparing association rate constants, BMP-2 binds faster to Noggin with higher stability, whereas BMP-9 exhibits a significantly weaker binding affinity. Immunoprecipitation followed by immunofluorescence studies demonstrated that BMP-9 binds to BMP type 2 receptor (BMPR2) regardless of Noggin binding, in contrast to BMP-2. To assess the impact of Noggin on BMP-2 and BMP-9 binding to BMPR2 in MC3T3-E1 cells, we performed immunoprecipitation and immunoblot analyses. In the absence of Noggin, both BMP-2 and BMP-9 effectively bound to BMPR2. However, in the presence of Noggin, BMP-2 binding to BMPR2 was inhibited. These results, supported by both computational and experimental analyses, highlight the potential of BMP-9 as a promising therapeutic agent for bone regeneration.