A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation

Abstract

The crystal structure of a collagen-binding domain (CBD) with an N-terminal domain linker from Clostridium histolyticum class I collagenase was determined at 1.00 A resolution in the absence of calcium (1NQJ) and at 1.65 A resolution in the presence of calcium (1NQD). The mature enzyme is composed of four domains: a metalloprotease domain, a spacing domain and two CBDs. A 12-residue-long linker is found at the N-terminus of each CBD. In the absence of calcium, the CBD reveals a beta-sheet sandwich fold with the linker adopting an alpha-helix. The addition of calcium unwinds the linker and anchors it to the distal side of the sandwich as a new beta-strand. The conformational change of the linker upon calcium binding is confirmed by changes in the Stokes and hydrodynamic radii as measured by size exclusion chromatography and by dynamic light scattering with and without calcium. Furthermore, extensive mutagenesis of conserved surface residues and collagen-binding studies allow us to identify the collagen-binding surface of the protein and propose likely collagen-protein binding models.

Unidirectional Binding of Clostridial Collagenase to Triple Helical Substrates

“…The native spectrum of CBD has only one-emission maxima at 317 nm. This comes from solvent-exposed tyrosine residues with the tryptophan being buried inside the core (no emission maxima at 350 nm) (6). The emission spectrum of CBD-collagenous peptide complex was identical to the native CBD spectrum, indicating that the tertiary structure of CBD does not change upon binding to tropocollagen (supplemental Fig.…”

“…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).…”

Discrimination between Distant Homologs and Structural Analogs: Lessons from Manually Constructed, Reliable Data Sets

“…In the all-β class, the highest-scoring pair (SVM score 7.59, probability 0.97) is the Clostridium histolyticum class I collagenase collagen-binding domain (CBD) 38 and the Clostridium stercorarium putative xylanase carbohydrate-binding module CBM6-3. 39 As shown in Fig.…”

Inhibitors of Clostridium histolyticum colagenase

“…The definition of which interactions are favorable to the inhibiting effect depends on the knowledge of which groups of the active site are important for the enzymatic activity. In the case of Clostridium histolyticum collagenase, Wilson et al [13] identified the amino acids residues relevant for the enzymatic activity, through the analysis of the mutagenesis effect of each residue upon such activity. The authors concluded that:…”