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.
Institut de Biologie et Chimie des Protéines, CNRS/Université de Lyon UMR 5086, IFR128 Biosciences Gerland Lyon Sud, 7, Passage du Vercors, 69367 Lyon Cedex 7, France.
Daniel
D
Kronenberg
Sandrine
S
Vadon-Le Goff
Jean-Marie
JM
Bourhis
Bernard
B
Font
Denise
D
Eichenberger
David J S
DJ
Hulmes
Catherine
C
Moali
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D055395:Bone Morphogenetic Protein 1 / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011347:Procollagen / Q000378:metabolism*; D011485:Protein Binding; D014162:Transfection
D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; C491555:PCOLCE protein, human; D011347:Procollagen; D055395:Bone Morphogenetic Protein 1