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 splicing variant of the gene encoding bone morphogenetic protein 1 (BMP-1). The variants have identical amino terminal signal peptides, prodomains and astacin-like protease domains. However, they differ in the length of their carboxy terminal part, which in pCP-2 has the composition CUB1, CUB2, EGF-like1, CUB3, EGF-like2, CUB4, CUB5, and C-tail. In the shorter form, pCP-1 (i.e., BMP-1), the sequence ends after the CUB3-domain. Using a combination of mutagenesis and structural approaches, we have investigated the structure and function of subfragments of pCP-2. The full-length latent recombinant enzyme and its N-terminally truncated form lacking the prodomain were tested for their enzymic activity. The intact protein showed only partial processing of procollagen type I, whereas the truncated form expressed enzymic activity 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 proteolysis of the recombinant protein with alpha-chymotrypsin produced four discrete fragments revealing the location of cleavage sites between the repetitive CUB/EGF domains. The results provide evidence that the CUB sequences form independently folded modules that are stabilized by two pairs of internal disulfide bridges. The modules are linked to each other by more flexible, hinge-like peptides. Solid-phase binding assays with isolated CUB domains and immobilized procollagen type I demonstrated that the first three but not the last two CUB domains specifically bound to the substrate. To define putative sites for CUB-CUB or CUB-substrate interactions, we generated molecular models for pCP-2 CUB domains. The models were obtained using as a template the structure of CUB domain in zona pellucida adhesion protein PSP-I/PSP-II from porcine sperm. The predicted conformations for homology models were, subsequently, confirmed by circular dichroism spectroscopy of polypeptide domains isolated following limited proteolysis with alpha-chymotrypsin.
Sieron
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AL
Center for Gene Therapy, Department of Biochemistry, MCP Hahnemann University, MCP Hahnemann School of Medicine, Philadelphia, Pennsylvania 19102, USA. Aleksander.Sieron@drexel.edu
Tretiakova
A
A
Jameson
B A
BA
Segall
M L
ML
Lund-Katz
S
S
Khan
M T
MT
Li
S w
Sw
Stöcker
W
W
D000595:Amino Acid Sequence; D000818:Animals; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002642:Chick Embryo; D002918:Chymotrypsin / Q000378:metabolism*; D002942:Circular Dichroism; D003198:Computer Simulation; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011347:Procollagen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D055394:Tolloid-Like Metalloproteinases
D019485:Bone Morphogenetic Proteins; D010446:Peptide Fragments; D011347:Procollagen; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D055394:Tolloid-Like Metalloproteinases; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D008666:Metalloendopeptidases; C485669:TLL2 protein, human; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1