Structure and function of procollagen C-proteinase (mTolloid) domains determined by protease digestion, circular dichroism, binding to procollagen type I, and computer modeling

Procollagen C-proteinase-2 (pCP-2, mTld) is derived from the longest splici ng variant of the gene encoding bone morphogenetic protein 1 (BMP-1), The v ariants have identical amino terminal signal peptides, prodomains and astac in-like protease domains. However, they differ in the length of their carbo xy terminal part, which in pCP-2 has the composition CUB1, CUB2, EGF-like1, CUB3, EGF-like2, CUB4, CUBS, and C-tail. In the shorter form, pCP-1 (i.e., BMP-I), the sequence ends after the CUB3-domain. Using a combination of mu tagenesis and structural approaches, we have investigated the structure and function of subfragments of pCP-2, The full-length latent recombinant enzy me and its N-terminally truncated form lacking the prodomain were tested fo r their enzymic activity. The intact protein showed only partial processing of procollagen type I, whereas the truncated form expressed enzymic activi ty indistinguishable from its native counterpart purified from chick embryo tendons. These results clearly demonstrated that the prodomain is required for the latency of the enzyme but not for its correct folding. Limited pro teolysis of the recombinant protein with alpha-chymotrypsin produced four d iscrete fragments revealing the location of cleavage sites between the repe titive CUB/EGF domains. The results provide evidence that the CUB sequences form independently folded modules that are stabilized by two pairs of inte rnal disulfide bridges, The modules are linked to each other by more flexib le, hinge-like peptides. Solid-phase binding assays with isolated CUB domai ns and immobilized procollagen type I demonstrated that the first three but not the last two CUB domains specifically bound to the substrate. To defin e putative sites for CUB-CUB or CUB-substrate interactions, we generated mo lecular models for pCP-2 CUB domains. The models were obtained using as a t emplate the structure of CUB domain in zona pellucida adhesion protein PSP- I/PSP-II from porcine sperm. The predicted conformations for homology model s were, subsequently, confirmed by circular dichroism spectroscopy of polyp eptide domains isolated following limited proteolysis with a-chymotrypsin.