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...

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Clostridium histolyticum collagenase is responsible for extensive tissue destruction in gas gangrene, and its activity is enhanced by calcium ions. The collagen‐binding domain is the minimal segment of the enzyme required for binding to insoluble collagen fibrils and for subsequent collagenolysis. The collagen‐binding domain is joined to another binding module by a conserved 14‐amino‐acid linker. The linker undergoes secondary structural transformation from an α‐helix to a β‐strand and forms a nonprolyl cis‐peptide in the presence of calcium ions. In this study, various biophysical methods were utilized to better understand the structure and functional role of the novel calcium‐activated linker. Two Ca2+ ions bind cooperatively with macroscopic association constants of K1 = 5.01 × 105 m−1 and K2 = 2.28 × 105 m−1. The chelation of the second calcium ion is enthalpically unfavorable, which could be a result of isomerization of the nonprolyl cis‐peptide. The holo protein is more stable than the apo protein against thermal denaturation (ΔTm ∼ 20 °C) and chemical denaturation (ΔΔGH2O ∼ 3 kcal·mol−1 for urea or guanidine HCl denaturation and Δ20% v/v in 2,2,2‐trifluoroethanol). The compact holo collagen‐binding domain is more resistant to proteolytic digestion than the apo collagen‐binding domain. The orientation of the linker appears to play a crucial role in the stability and dynamics of the collagen‐binding domain.

Clostridium histolyticum collagenase causes extensive degradation of collagen in connective tissue that results in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to a collagen fibril. CBD binds unidirectionally to the undertwisted C-terminus of triple helical collagen. Here, we examine whether CBD could also target undertwisted regions even in the middle of the triple helix. Collageneous peptides with an additional undertwisted region were synthesized by introducing a Gly fi Ala substitution [(POG) x POA(POG) y ] 3 , where x 1 y 5 9 and x > 3). CBD binds to either the Gly fi Ala substitution site or to the C-terminus of each minicollagen. Smallangle X-ray scattering measurements revealed that CBD prefers to bind the Gly fi Ala site to the C-terminus. The HSQC NMR spectra of 15 N-labeled minicollagen and minicollagen with undertwisted regions were unaffected by the titration of unlabeled CBD. The results imply that CBD binds to the undertwisted region of the minicollagen but does not actively unwind the triple helix.

Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.