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.
Blanc
Guillaume
G
Institut de Biologie et Chimie des Protéines, CNRS/Université Claude Bernard Lyon 1, Unité Mixte de Recherche 5086, Institut Fédératif de Recherche 128 Biosciences Lyon Gerland, Lyon cedex 7, France.
Font
Bernard
B
Eichenberger
Denise
D
Moreau
Christophe
C
Ricard-Blum
Sylvie
S
Hulmes
David J S
DJ
Moali
Catherine
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D017124:Conserved Sequence; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D005453:Fluorescence; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D008666:Metalloendopeptidases; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1