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The Crystal Structure of the BMP-2:BMPR-IA Complex and the Generation of BMP-2 Antagonists

Investigation performed at Physiologische Chemie II, Biozentrum der Universität Würzburg, Würzburg, Germany
Joachim Nickel, PhD
Matthias K. Dreyer, PhD
Walter Sebald, PhD
Physiologische Chemie II, Biozentrum der Universität Würzburg, Am Hubland, 97074 Würzburg, Germany. E-mail address for W. Sebald: sebald{at}biozentrum.uni-wuerzburg.de
Thomas Kirsch, PhD
Medigene, Lochhamer Str. 11, 82152 Martinsried, Germany
In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Deutsche Forschungsgemeinschaft (DFG) Grant SFB 487 TP B1 and B2. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

Background: Bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) belong to the large transforming growth factor-ß (TGF-ß) superfamily of multifunctional cytokines. Signaling of the BMPs requires 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-ß superfamily exhibit stringent specificity in their ligand-receptor interactions, which may be a reason for the qualitative and quantitative differences in cellular responses. To understand how BMPs and GDFs activate their receptors, it is important to determine structure and binding mechanisms of ligand-receptor complexes. We have used BMP-2 as a key representative of the BMPs to identify the epitopes for type I and type II receptor binding by mutational interaction analyses and have solved the crystal structure of a BMP-2:BMPR-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 subjected to interaction analyses with use of the receptor ectodomains of BMPR-IA, BMPR-II, and ActR-II immobilized on a biosensor system. The biological activity of the BMP-2 variants was measured by BMP-2 dependent expression of alkaline phosphatase (ALP) in C2C12 cells. For crystallization, a complex 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 identified distinct epitopes for type I and type II receptor binding. Because the structure of TGF-ß-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:BMPR-IA ectodomain complex revealed a key feature of the ligand-receptor interaction: a large hydrophobic residue (Phe85) within a hydrophobic patch of BMPR-IA fit into a hydrophobic pocket composed of residues of both BMP-2 monomers. A second epitope identified by alanine mutagenesis scanning was termed 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 the knuckle epitope produced variants with altered biological activities. Variants 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 similarities between members of the TGF-ß superfamily, it can be assumed that the data presented in this work are transferable to other TGF-ß receptor systems. Because of the association with various diseases, the generation of antagonists of other TGF-ß superfamily members might generate potent tools for basic research and therapeutic approaches.

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