Unidirectional Binding of Clostridial Collagenase to Triple Helical Substrates

Abstract

Histotoxic clostridia produce collagenases responsible for extensive tissue destruction in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to collagen fibril. Collagen binding efficiency of CBD is more pronounced in the presence of Ca Histotoxic clostridia produce collagenases that degrade collagen in connective tissue. Although the enzyme is assumed to be a causative agent for diseases like gas gangrene (1), it is beneficial to remove dead tissue from ulcers or burns and for nonsurgical treatment of Dupuytren's disease (2, 3). 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. 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).Since collagen fibrils constitute a major part of the extracellular matrix, bioactive molecules can be anchored with CBD for their prolonged effect. Nishi et al. (7) have demonstrated that growth factors fused to CBD remained at the sites of injection much longer than growth factors alone to induce extended cell proliferation. In order to gain an insight into the anchoring mechanism of CBD, we attempted to co-crystallize CBD and collagenous peptide without success. Also to better address the role of CBD in fibril disruption and transition states from insoluble substrate, solution studies of CBD with the triple helical collagenous peptide became necessary.NMR titration methods were utilized to identify the collagen binding pocket on CBD. Since it has been shown that most peptidases bind to their substrate in one direction at their catalytic center (8, 9), there could be only one direction for the collagen triple helices at the binding site of CBD. On the other hand, CBD might allow bidirectional binding, since it is independent of the catalytic domain. To identify the binding direction, three different NMR titrations were performed with spinlabeled analogues of tropocollagen, where a nitroxide spin label 2,2,5,5-tetramethyl-L-pyrrolidinyloxy (PROXYL) was attached to either the N or C terminus of the collagenous peptide. The nitroxide moiety with an unpaired electron can cause enhancement in pa...

Structural Comparison of ColH and ColG Collagen-Binding Domains from Clostridium histolyticum

“…The crystal structures of s3 and s3b are most divergent at the N terminus and the loops connecting the ␤-strands. For example, a loop of 8 residues (Glu961-Ile968) between ␤-strands E and F in s3b are line broadened and nonassignable on NMR, and the flexible loop is unobserved in the crystal structure (10,11). In contrast, the equivalent loop in s3 (Glu930-Val935) is well ordered.…”

“…complex with collagenous peptides showed that Ser928 and Arg929 interact with the C terminus of collagenous peptide (10). Neither residue is conserved in s3.…”

“…4). Our previous work demonstrated that both (POG) 10 (L-hydroxyproline is represented as O) and G(POG) 7 PRG adopt a triple-helical structure in solution. They are thus represented as [(POG) 10 ] 3 and [G(POG) 7 PRG] 3 , respectively.…”

“…The collagen-binding surface in s3b was identified by binding assay of 32 surface mutants (11) and by HSQC-NMR titration (10). Three aromatic side chains critically involved in collagen binding are positioned similarly in s3.…”

“…The s3b binds to collagenous peptides with triple-helical conformation but not to the similar peptides lacking triple-helical conformation or to gelatin (denatured collagen), suggesting that the CBD-collagen interaction is conformation specific (4,7). The most recent solution studies of s3b in complex with collagenous peptides by smallangle X-ray scattering (SAXS) and heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) titration of various spin-labeled collagenous peptides to 15 N-labeled s3b showed that s3b binds unidirectionally to the C terminus of collagenous peptides (10).…”