Substrate Recognition by the Collagen-binding Domain ofClostridium 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-HypGly) 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 triplehelical 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.Collagens are the major components of the extracellular matrix. They are the most abundant proteins in mammals, constituting a quarter of their total weight. Collagens are not only structural proteins with a high tensile strength but also affect cell differentiation, migration, and attachment. Nineteen different types are known to date, and type I collagen is the major species in higher vertebrates. In the endoplasmic reticulum of fibroblasts, collagen precursor peptides are hydroxylated by various dioxygenases (prolyl hydroxylases and a lysyl hydroxylase), glycosylated at the hydroxylysine residues, and folded into a triple helix. HSP47 associates with procollagen during this modification and/or folding processes and is assumed to function as a collagen-specific molecular chaperone (1). The precursor is secreted into the extracellular space, and its N-and C-terminal propeptides are removed by procollagen peptidases. Cleavage triggers the arrangement of collagen monomers into a staggered array called fibrils, which are insoluble macromolecular assemblies having lengths up to a few hundred micrometers. One can observe 67-nm-wide cross striations (overlap and gap zones) on the fibrils by electron microscopy, which are formed by the regular arrangement of collagen monomers. Each collagen monomer consists of collagenous and noncollagenous regions. The former have a regular amino acid sequence, which is a repeat (338 times in type I collagen) of the Gly-X-Y triplet, forming a long (300 nm in type I collagen) triple-helical domain. Proline and hydroxyproline residues are most common in the X and Y positions, respectively, and this combination stabilizes the helix to the highest extent (2). The latter regions lack the sequence and conformation characteristic of the former, and are named telopeptides,...

Unidirectional Binding of Clostridial Collagenase to Triple Helical Substrates

“…Each CBD spans ϳ120 amino acid residues and binds specifically to insoluble collagen. CBD also binds to collagenous peptides with triple helical conformation but not to collagenous peptides that lack triple helix or to gelatin (denatured collagen), suggesting that the CBD-collagen interaction is conformation-specific (4,5). Calcium ions enhance the binding at physiological concentration, and x-ray crystal structures of CBD have been solved in the presence and absence of calcium (6).…”

“…For collagenases to hydrolyze tissue collagen, the enzymes must 1) anchor themselves onto an insoluble collagen fibril, which is a staggered array of tropocollagen and then 2) isolate a single triple helical molecule from the bundle and finally 3) unwind the triple helix to expose a scissile peptide bond. Clostridium histolyticum produces two classes of collagenases, which contain a catalytic domain belonging to the family M9B, followed by one or two copies of polycystic kidney disease domains and one or two copies of collagen-binding domains (CBD) 2 (4). Each CBD spans ϳ120 amino acid residues and binds specifically to insoluble collagen.…”

“…The glutathione S-transferase tag was cleaved off by thrombin, and CBD was purified as described previously (4). Uniform 15 N isotope labeling was achieved using Tanaka minimal medium containing 40 mM 15 NH 4 Cl.…”

The FAXWXXT motif in the carboxyl terminus of Vibrio mimicus metalloprotease is involved in binding to collagen

“…S3 in class I collagenase forms tandem repeating structure, S3a and S3b, and showed high homology between these segments. S3a and S3b had respective collagen binding activities and addition of S3a to S3b exhibited the most efficient binding to collagen [29]. In contrast to class I collagenase, S3 fragment in class II collagenase had only one CBD.…”

Constructed amphiphilic gel as natural auxiliary medium to mimick remineralized dental hydroxyapatite

“…The calcification solution pH was adjusted by 1 M HCl to 7.60 using a Metrohm 718 pH-STAT. [13]. All the samples were studied by X-ray diffraction (XRD, X'Pert Pro MPD, Philips, Holland), SEM.…”