The crystal structure of the BMP-2 : BMPR-IA complex and the generation ofBMP-2 antagonists

Background: Bone morphogenetic proteins (BMPs) and growth and differentiati on factors (GDFs) belong to the large transforming growth factor-beta (TGF- beta) superfamily of multifunctional cytokines. Signaling of the BMPs requi res the binding of the BMP to the BMP cell surface receptors BMPR-IA, BMPR- IB, and BMPR-II. Similar to other cytokines, members of the TGF-beta superf amily exhibit stringent specificity in their ligand-receptor interactions, which may be a reason for the qualitative and quantitative differences in c ellular responses. To understand how BMPs and GDFs activate their receptors , it is important to determine structure and binding mechanisms of ligand-r eceptor complexes. We have used BMP-2 as a key representative of the BMPs t o identify the epitopes for type I and type II receptor binding by mutation al interaction analyses and have solved the crystal structure of a BMP-2:BM PR-IA receptor ectodomain complex. Methods: To identify amino acid side chains involved in receptor binding, a collection of in vitro mutagenized human BMP-2 variants was prepared and s ubjected to interaction analyses with use of the receptor ectodomains of BM PR-IA, BMPR-II, and ActR-Il immobilized on a biosensor system. The biologic al activity of the BMP-2 variants was measured by BMP-2 dependent expressio n of alkaline phosphatase (ALP) in C2C12 cells. For crystallization, a comp lex of BMP-2 and the ectodomain of BMPR-IA was formed in solution, purified , and crystallized as described(12). Results: The ligand-receptor interaction analysis of the BMP-2 variants ide ntified distinct epitopes for type I and type II receptor binding. Because the structure of TGF-beta -like proteins has been compared with that of an open hand, the binding epitope for the type I receptor was-on the basis of its location-termed "wrist" epitope. The crystal structure of the BMP-2:BMP R-IA ectodomain complex revealed a key feature of the ligand-receptor inter action: a large hydrophobic residue (Phe85) within a hydrophobic patch of B MPR-IA fit into a hydrophobic pocket composed of residues of both BMP-2 mon omers. A second epitope identified by alanine mutagenesis scanning was term ed the "knuckle" epitope on the basis of its location on the outer side of the "finger" segments of BMP-2. Mutations in either the wrist epitope or th e knuckle epitope produced variants with altered biological activities. Var iants with antagonistic properties were exclusively generated by mutations in the knuckle epitope of BMP-2. Conclusions and Clinical Relevance: The identification and characterization of the two receptor binding epitopes in BMP-2 provide new insight into the primary steps of BMP-receptor activation. Because of the structural simila rities between members of the TGF-beta superfamily, it can be assumed that the data presented in this work are transferable to other TGF-beta receptor systems. Because of the association with various diseases, the generation of antagonists of other TGF-beta superfamily members might generate potent tools for basic research and therapeutic approaches.