ABSTRACT
The bone morphogenetic protein (BMP)-1/tolloid metalloproteinases are evolutionarily conserved enzymes that are fundamental to dorsal-ventral patterning and tissue morphogenesis. The lack of knowledge regarding how these proteinases recognize and cleave their substrates represents a major hurdle to understanding tissue assembly and embryonic patterning. Although BMP-1 and mammalian tolloid (mTLD) are splice variants, it is puzzling why BMP-1, which lacks 3 of the 7 noncatalytic domains present in all other family members, is the most effective proteinase. Using a combination of single-particle electron microscopy, small-angle X-ray scattering, and other biophysical measurements in solution, we show that mTLD, but not BMP-1, forms a calcium-ion-dependent dimer under physiological conditions. Using a domain deletion approach, we provide evidence that EGF2, which is absent in BMP-1, is critical to the formation of the dimer. Based on a combination of structural and functional data, we propose that mTLD activity is regulated by a substrate exclusion mechanism. These results provide a mechanistic insight into how alternative splicing of the Bmp1 gene produces 2 proteinases with differing biological activities and have broad implications for regulation of BMP-1/mTLD and related proteinases during BMP signaling and tissue assembly.
Subject(s)
Protein Multimerization , Tolloid-Like Metalloproteinases/chemistry , Tolloid-Like Metalloproteinases/metabolism , Animals , Calcium/metabolism , Cell Line , Computer Simulation , Humans , Microscopy, Electron, Transmission , Models, Molecular , Protein Structure, Quaternary , Protein Structure, Tertiary , Structural Homology, Protein , Substrate Specificity , Tolloid-Like Metalloproteinases/genetics , Tolloid-Like Metalloproteinases/ultrastructureABSTRACT
Bone morphogenetic protein-1 (BMP-1)/tolloid proteinases are fundamental to regulating dorsal ventral patterning and extracellular matrix deposition. In mammals there are four proteinases, the splice variants BMP-1 and mammalian tolloid (mTLD), and tolloid like-1 and -2 (TLL-1/2). BMP-1 has the highest catalytic activity and lacks three non-catalytic domains. We demonstrate that TLL-1, which has intermediate activity, forms a calcium-ion dependent dimer with monomers stacked side-by-side. In contrast, truncated TLL-1 molecules having the same shorter structure as BMP-1 are monomers and have improved activity towards their substrate chordin. The increased activity exceeds not only that of full-length TLL-1 but also BMP-1.