Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase

Clostridium histolyticum type I collagenase (ColG) has a segmental structur e, 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 S3aS3b showed the most efficient binding to substrate, cooperative binding by both domains was suggested for the enzyme. Monomeric (S3b) and tandem (S3aS3b) CBDs bound to atelocollagen, which contains only the collagenous regio n. However, they did not bind to telopeptides immobilized on Sepharose bead s. These results suggested that the binding site(s) for the CBD is(are) pre sent in the collagenous region. The CBD bound to immobilized collagenous pe ptides, (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 b ind to various peptides with similar amino acid sequences or to gelatin, wh ich lacks a triple-helical conformation, The CBD did not bind to immobilize d Glc-Gal disaccharide, which is attached to the side chains of hydroxylysi ne residues in the collagenous region. These observations suggested that th e CBD specifically recognizes the triple helical conformation made by three polypeptide chains in the collagenous region.