Basic research on the development of novel vaccines with collagen-anchoring potency.

点击翻译按钮获取中文摘要

The crystal structure of a collagen-binding domain with an N-terminal domain linker from Clostridium histolyticum class I collagenase was determined in the absence and presence of calcium. The mature enzyme is composed of four domains, a catalytic domain, a spacing domain (PKD), and two collagen-binding domains (CBDs). The CBD monomer reveals a beta-sheet sandwich fold. Extensive mutagenesis of conserved surface residues and collagen-binding studies allow us to identify the protein's collagen-binding surface and propose likely collagen-protein binding models. A twelve-residue-long linker is found at the N-terminus of each CBD. In the absence of calcium, the linker adopts 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. The domain becomes more rigid and efficient for collagen-binding in the presence of calcium.In addition, various collagenases were purified from other Clostridial species. Their structural genes were sequenced to show that they possess variable domain organizations. These implies reiterated domain-duplication events during gene evolution. In order to reveal molecular basis to hydrolyze triple-helical peptide substrates, we have produced recombinant catalytic domains derived from these enzymes. We started their structural analysis by X-ray crystallography. At the moment, crystals were obtained from C. histolyticum class I collagenase, which are under the X-ray analysis.