title: Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase.
abstract: Clostridium histolyticum type I collagenase (ColG) has a segmental structure, S1+S2+S3a+S3b. S3a and S3b bound to insoluble collagen, but S2 did not, thus indicating that S3 forms a collagen-binding domain (CBD). Because S3a+S3b showed the most efficient binding to substrate, cooperative binding by both domains was suggested for the enzyme. Monomeric (S3b) and tandem (S3a+S3b) CBDs bound to atelocollagen, which contains only the collagenous region. However, they did not bind to telopeptides immobilized on Sepharose beads. These results suggested that the binding site(s) for the CBD is(are) present in the collagenous region. The CBD bound to immobilized collagenous peptides, (Pro-Hyp-Gly)(n) and (Pro-Pro-Gly)(n), only when n is large enough to allow the peptides to have a triple-helical conformation. They did not bind to various peptides with similar amino acid sequences or to gelatin, which lacks a triple-helical conformation. The CBD did not bind to immobilized Glc-Gal disaccharide, which is attached to the side chains of hydroxylysine residues in the collagenous region. These observations suggested that the CBD specifically recognizes the triple-helical conformation made by three polypeptide chains in the collagenous region.
authors: Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. microbio@kms.ac.jp
authors: O
authors: O
authors: Matsushita
authors: T
authors: T
authors: Koide
authors: R
authors: R
authors: Kobayashi
authors: K
authors: K
authors: Nagata
authors: A
authors: A
authors: Okabe
mesh_terms: D001483:Base Sequence; D003013:Clostridium / Q000201:enzymology*; D003094:Collagen / Q000378:metabolism*; D017364:Collagenases / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
chemical_list: D017931:DNA Primers; D003094:Collagen; D017364:Collagenases