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

Abstract

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...

Strong Cooperativity and Loose Geometry between CUB Domains Are the Basis for Procollagen C-Proteinase Enhancer Activity

“…Interestingly, the CUB domain region alone is sufficient to promote enhancement (4,10). We have shown that the triple helix of procollagens is not required for tolloid stimulation (6) and have identified several residues in CUB1 that seem to play a major role in the PCPE-procollagen interaction (11). Of note, when the calcium-binding site in CUB1 is disrupted, interaction with the C-terminal part of procollagen III is completely abolished, * This work was supported by the Ré gion Rhô ne-Alpes, the European Com-locating one possible interaction site on loops 5, 7, and 9 of CUB1.…”

“…1A). CUB1NTR and CUB2NTR were produced with the QuikChange XL site-directed mutagenesis kit from Stratagene using the cDNA of human PCPE-1 inserted into the pCEP4 vector (Invitrogen), in-frame with an 8-histidine C-terminal tag (known as PCPEhis) (11). 5Ј-Phosphorylated primers were designed to delete residues 150 -275 to obtain CUB1NTR (numbering according to human full-length PCPE-1, including the signal peptide) and residues 26 -149 to obtain CUB2NTR.…”

Regulation of apoAI processing by procollagen C-proteinase enhancer-2 and bone morphogenetic protein-1

“…In addition to high homology to its family member PCPE1, homology with PCPE2 was found with several proteins containing CUB domains including the protein encoded by the Cubulin gene, BMP-1, and other members of the astacin family (14). The two CUB domains near the N terminus are the most conserved regions of the protein with the C-terminal NTR domain less well conserved.…”

“…The cDNA encodes a 415-amino acid protein that has 43% identity to the type I procollagen C-proteinase enhancer protein (PCPE1) (13). PCPE2, like PCPE1, contains two CUB (complement C1r/C1s, Uegf, Bmp1) domains, which are thought to be important in protein-protein interaction (14) and binding to the COOH-terminal propeptide of type I procollagen leading to the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1 (BMP-1) (13). In addition, the recent finding identifying BMP-1 as the key enzyme responsible for the cleavage of pro-apoAI into its mature form (15) raises the intriguing possibility that PCPE2 alone or in combination with BMP-1 participates in the processing of proapoAI and therefore influence the biogenesis of HDL.…”

Metalloproteinases in Drosophila to Humans That Are Central Players in Developmental Processes

“…A subset of CUB domains appears to require Ca 2ϩ for optimum binding activity (16). The most N-terminal BMP1 CUB domain (C1) may play a role in imparting "chordinase" activity, or ability to cleave chordin (17), a substrate described below.…”