Strong cooperativity and loose geometry between CUB domains are the basis for procollagen c-proteinase enhancer activity.

Procollagen C-proteinase enhancers (PCPE-1 and -2) specifically activate bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family during C-terminal processing of fibrillar collagen precursors. PCPEs consist of two CUB domains (CUB1 and CUB2) and one NTR domain separated by one short and one long linker. It was previously shown that PCPEs can strongly interact with procollagen molecules, but the exact mechanism by which they enhance BMP-1 activity remains largely unknown. Here, we used a series of deletion mutants of PCPE-1 and two chimeric constructs with repetitions of the same CUB domain to study the role of each domain and linker. Out of all the forms tested, only those containing both CUB1 and CUB2 were capable of enhancing BMP-1 activity and binding to a mini-procollagen substrate with nanomolar affinity. Both these properties were lost by individual CUB domains, which had dissociation constants at least three orders of magnitude higher. In addition, none of the constructs tested could inhibit PCPE activity, although CUB2CUB2NTR was found to modulate BMP-1 activity through direct complex formation with the enzyme, resulting in a decreased rate of substrate processing. Finally, increasing the length of the short linker between CUB1 and CUB2 was without detrimental effect on both activity and substrate binding. These data support the conclusion that CUB1 and CUB2 bind to the procollagen substrate in a cooperative manner, involving the short linker that provides a flexible tether linking the two binding regions.

Insights into how CUB domains can exert specific functions while sharing a common fold: conserved and specific features of the CUB1 domain contribute to the molecular basis of procollagen C-proteinase enhancer-1 activity.

Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies.

Substrate-specific modulation of a multisubstrate proteinase. C-terminal processing of fibrillar procollagens is the only BMP-1-dependent activity to be enhanced by PCPE-1.

Members of the bone morphogenetic protein-1/tolloid (BMP-1/Tld) family of metalloproteinases, also known as procollagen C-proteinases (PCPs), control multiple biological events (including matrix assembly, cross-linking, cell adhesion/migration and pattern formation) through enzymatic processing of several extracellular substrates. PCP activities on fibrillar procollagens can be stimulated by another family of extracellular proteins, PCP enhancers (PCPE-1, PCPE-2), which lack intrinsic enzymatic activity. While PCPs have multiple substrates, the extent to which PCPEs is involved in the processing of proteins other than fibrillar procollagens is unknown. In the experiments reported here, PCPE-1 was found to have no effect on the in vitro BMP-1 processing of procollagen VII, the procollagen V N-propeptide, the laminin 5 gamma2 chain, osteoglycin, prolysyl oxidase, or chordin. In contrast, PCPE-1 enhanced C-terminal processing of human fibrillar procollagen III but only when this substrate was in its native, disulfide-bonded conformation. Surprisingly, processing of procollagen III continued to be enhanced when essentially all the triple-helical region was removed. These and previous results (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869; Bernocco, S., Steiglitz, B. M., Svergun, D. I., Petoukhov, M. V., Ruggiero, F., Ricard-Blum, S., Ebel, C., Geourjon, C., Deleage, G., Font, B., Eichenberger, D., Greenspan, D. S., and Hulmes, D. J. S. (2003) J. Biol. Chem. 278, 7199-7205) indicate that the mechanism of PCPE-1 action involves recognition sites in both the C-propeptide domain and in the C-telopeptide region of the procollagen molecule. PCPEs therefore define a new class of extracellular adaptor proteins that stimulate proteinase activity in a substrate-specific manner, thereby providing a new target for the selective regulation of PCP activity on fibrillar procollagen substrates.

Low resolution structure determination shows procollagen C-proteinase enhancer to be an elongated multidomain glycoprotein.

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that can stimulate the action of tolloid metalloproteinases, such as bone morphogenetic protein-1, on a procollagen substrate, by up to 20-fold. The PCPE molecule consists of two CUB domains followed by a C-terminal NTR (netrin-like) domain. In order to obtain structural insights into the function of PCPE, the recombinant protein was characterized by a range of biophysical techniques, including analytical ultracentrifugation, transmission electron microscopy, and small angle x-ray scattering. All three approaches showed PCPE to be a rod-like molecule, with a length of approximately 150 A. Homology modeling of both CUB domains and the NTR domain was consistent with the low-resolution structure of PCPE deduced from the small angle x-ray scattering data. Comparison with the low-resolution structure of the procollagen C-terminal region supports a recently proposed model (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869) for the mechanism of action of PCPE.

Interaction properties of the procollagen C-proteinase enhancer protein shed light on the mechanism of stimulation of BMP-1.

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150-400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.