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

在无钙和有钙存在的情况下，测定了溶组梭菌I型胶原酶N-末端结构域连接物的胶原结合域的晶体结构。成熟的酶由四个结构域组成，一个催化域、一个间隔域(PKD)和两个胶原结合域(CBD)。CBD单体显示出贝塔折叠的夹心层。对保守的表面残基的广泛突变和胶原结合的研究使我们能够确定蛋白质的胶原结合表面，并提出可能的胶原-蛋白质结合模型。在每个CBD的N-末端发现一个12个残基的长接头。在没有钙的情况下，连接子采用阿尔法螺旋。添加的钙解开连接子，并将其锚定在三明治的远端，作为新的β-链。结合钙时连接物的构象变化被尺寸排阻色谱测量的斯托克斯半径和流体动力学半径的变化以及有无钙的动态光散射所证实。在有钙存在的情况下，结构域变得更刚性和更有效地与胶原结合。此外，从其他梭状芽胞杆菌中纯化了各种胶原酶。对它们的结构基因进行了测序，表明它们具有可变域组织。这意味着在基因进化过程中重复发生了结构域复制事件。为了揭示水解三螺旋多肽底物的分子基础，我们制作了由这些酶衍生的重组催化结构域。我们开始用X射线结晶学对它们进行结构分析。目前，在X射线分析下得到了溶组链球菌I类胶原酶的晶体。

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.