X-ray crystallographic and biochemical studies on the bone morphogenetic protein family

Bone Morphogenetic Proteins (BMPs) are extracellular messenger ligands involved in controlling a wide array of developmental and intercellular signaling processes. To initiate their specific intracellular signaling pathways, the ligands recognize and bind two structurally related serine/threonine kinase receptors, termed type I and type II, on the cell surface. To address the structural arrangement of the receptors when bound to the ligand, the structure of BMP-2, ligand, bound to its type I receptor BMPRIa-ECD and type II receptor ActRII-ECD was determined. The structural arrangement this complete, signaling competent complex confirms that the two receptor types do not directly contact each other. Further, comparison of previously solved high affinity type II receptor/ligand interfaces with the lower affinity interface of BMP-2/ ActRII-ECD allowed for identification of ligand residues important for determining receptor affinity. A known feature of BMP complex assembly is the cooperative nature of receptor binding. When bound to its high affinity receptor, the ligand's affinity for the lower affinity receptor is increased. However, the lack of conformational changes to either the receptors or ligand in the ternary complex leaves the mechanism unclear. Using the natural homo/hetero-dimer system of activin/Inihibin, the nature of this cooperativity was probed. Activin's receptor affinity was shown to vary depending on the surface concentration of the receptor, whereas Inhibin's receptor affinity remained constant. This finding suggests cooperative receptor binding is a result of increased local concentration and loss of rotational freedom of the ligand upon binding to a high affinity receptor. Finally, structural and biochemical studies were undertaken for two new BMP ligands, BMP-3 and BMP-6. Interestingly, while BMP -6 exhibited many similarities to BMP-7, BMP-3 displayed a previously unseen 30-fold specificity difference between ActRIIb-ECD and ActRII-ECD. Comparison of the predicted interfaces of these receptors with BMP-3 yielded a single residue interaction which regulates this receptor preference. The combination of these related studies illustrates how single amino acid differences between ligands can effect receptor binding and, ultimately, impact BMP signaling and functio