Biophysical Characterization of the C-propeptide Trimer from Human Procollagen III Reveals a Tri-lobed Structure

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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 threedimensional 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. CUB3 domains are widely occurring structural motifs, found almost exclusively in extracellular and plasma membrane-associated proteins. These proteins are involved in a wide range of biological functions, including complement activation (1, 2), developmental patterning (3, 4), tissue repair (5), axon guidance and angiogenesis (6, 7), cell signaling (8), fertilization (9), hemostasis (10), inflammation (11), neurotransmission (12), receptor-mediated endocytosis (13,14), and tumor suppression (15, 16). Many CUB domain-containing proteins are proteases (1,2,5,10,(17)(18)(19). Although the roles of the CUB domains are largely unexplored, a number of them have been shown to be involved in oligomerization and/or recognition of substrates and binding partners.The protein families from which the CUB domain derives its name are the complement serine proteases C1r, C1s, MASP-1, MASP-2, and MASP-3 and the bone morphogenetic protein-1/ tolloid metalloproteases BMP-1, mTLD, mTLL-1, and mTLL-2 (or their counterparts xolloids, tolloids, and SpAN/BP10 in Xenopus, Drosophila, and sea urchin, respectively). Each consists of a catalytic domain (either N-terminal in tolloids or C-terminal in complement proteases) together with several CUB domains interspersed by calcium-binding EGF domains. In the case of the complement proteases, the CUB domain...

Section: Discussionmentioning

confidence: 90%

Insights into How CUB Domains Can Exert Specific Functions while Sharing a Common Fold

Blanc

Font

Eichenberger

et al. 2007

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“…Proteins and Antibodies-Recombinant human PCPE as well as the C-terminal propeptide trimer from procollagen III (CPIII) were expressed using baculovirus systems (19,37,38). Procollagen I and its C-propeptide trimer domain (CPI) were purified from the culture media of chick embryo tendon fibroblasts (39) or chick embryo tendons (40), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Because relatively weak calcium binding can also be attributed to glutamic acid-or aspartic acid-rich sequences (50,51), these also may represent potential Ca 2ϩ binding sites. Possible interaction sites for PCPE in the procollagen Cpropeptide region can be proposed on the basis of recent small angle x-ray scattering studies of CPIII (37). These indicate a structure consisting of three major lobes, each of which might correspond to the C-terminal region of each component chain containing the internal disulfide bonds, plus one minor lobe corresponding to a putative N-terminal junction region (residues 1-80) containing the interchain disulfide bonds.…”

Section: Figmentioning

confidence: 99%

Interaction Properties of the Procollagen C-proteinase Enhancer Protein Shed Light on the Mechanism of Stimulation of BMP-1

Ricard‐Blum

Bernocco

Font

et al. 2002

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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 Cpropeptide 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.Bone morphogenetic protein-1 (BMP-1) 1 and other tolloidrelated metalloproteinases (1, 2), also known as procollagen C-proteinases (PCPs), have recently been shown to be involved in the control of a variety of morphogenetic events during development and tissue repair. These include: (i) the deposition of collagen fibrils in the extracellular matrix following the processing of procollagen propeptides (3-6); (ii) dorsoventral patterning (7-10) through the cleavage of the growth factor inhibitors chordin and SOG; (iii) collagen and elastin crosslinking by the processing of the inactive precursors of lysyl oxidases (11, 12); and (iv) adhesion/migration of epithelial cells by the cleavage of laminin 5 chains (13-15). The activities of PCPs on procollagen substrates may be stimulated up to 20-fold by another glycoprotein of the extracellular matrix, procollagen C-proteinase enhancer (PCPE) (16 -19), which lacks intrinsic proteinase activity. Similarly, in the case of chordin and SOG, the protein TSG or its homologues stimulates cleavage by tolloid proteinases (20, 21), thus raising the possibility that PCP processing of different substrates might be specifically regulated by distinct enhancer proteins.Both tolloid proteinases and PCPE are multidomain glycoproteins containing multiple copies of the so-called CUB domain (22), a protein module common to several extracellular and plasma membrane-associated...

“…Recent studies using low angle x-ray scattering have shown that the low resolution structure of the isolated C-propeptide trimer from type III procollagen consists of three major lobes together with one minor lobe (59). We hypothesized that each major lobe corresponds to the loop containing the intra-chain disulfide bonds in each of the three component chains, whereas the minor lobe corresponds to a putative junction region.…”

Section: C-terminal Coiled Coils In Other Members Of the Collagenmentioning

confidence: 99%

α-Helical Coiled-coil Oligomerization Domains Are Almost Ubiquitous in the Collagen Superfamily

McAlinden

Smith

Sandell

et al. 2003

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␣-Helical coiled coils are widely occurring protein oligomerization motifs. Here we show that most members of the collagen superfamily contain short, repeating heptad sequences typical of coiled coils. Such sequences are found at the N-terminal ends of the C-propeptide domains in all fibrillar procollagens. When fused C-terminal to a reporter molecule containing a collagen-like sequence that does not spontaneously trimerize, the Cpropeptide heptad repeats induced trimerization. C-terminal heptad repeats were also found in the oligomerization domains of the multiplexins (collagens XV and XVIII). N-terminal heptad repeats are known to drive trimerization in transmembrane collagens, whereas fibril-associated collagens with interrupted triple helices, as well as collagens VII, XIII, XXIII, and XXV, were found to contain heptad repeats between collagen domains. Finally, heptad repeats were found in the von Willebrand factor A domains known to be involved in trimerization of collagen VI, as well as in collagen VII. These observations suggest that coiled-coil oligomerization domains are widely used in the assembly of collagens and collagen-like proteins.The mechanisms controlling chain oligomerization in extracellular matrix and related proteins are currently topics of active research (1, 2). In the case of the collagen superfamily, which includes both collagens and collagen-like proteins (3-5), a number of structural features have been identified that are essential for bringing together component polypeptide chains with the correct stoichiometry, thus leading to folding of the triple helix. Early studies on the procollagen precursors of the fibrillar collagens (types I-III, V, and XI) (6) showed that the C-propeptide regions are necessary to direct correct chain association, which is followed by zipper-like folding of the triple helix in the C-to N-terminal direction (7). This concept was subsequently extended to basement membrane collagen type IV (8 -10), microfibril-forming collagen VI (11, 12), members of the C1q family (13) including collagens VIII and X (14 -17) and the emilins (18), and the FACITs 1 (19 -21).␣-Helical coiled coils have been shown to be oligomerization domains in both collagens and collagen-like proteins. The paradigm here is the collectin family (22), which includes the lung surfactant proteins (SP-A and SP-D), mannan-binding proteins (MBP-A and MBP-C), conglutinin, and collectin-43. Collectins are trimeric molecules in which each chain contains a collagenlike domain followed by an ␣-helical coiled-coil region and then a carbohydrate recognition domain (CRD). The amino acid sequence of the coiled-coil domain is characterized by up to four heptad repeats (a-b-c-d-e-f-g) in which hydrophobic amino acid residues occur at positions a and d (23). Three-dimensional structures of the coiled-coil and CRD regions in MBP and SP-D show that chains within the trimer are associated via a threestranded coiled coil, with the three CRDs arranged as distinct lobes (24 -26). Several studies have shown that the co...