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

具有胶原锚定功效的新型疫苗开发的基础研究。

基本信息

批准号：
14570239
负责人：
MATSUSHITA Osamu
金额：
$ 2.3万
依托单位：
Kagawa University(Faculty of Medicine)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

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.

在不存在和存在钙的情况下，测定了来自溶组织梭菌I类胶原酶的具有N-末端结构域接头的胶原结合结构域的晶体结构。成熟的酶由四个结构域组成，催化结构域，间隔结构域（PKD）和两个胶原结合结构域（CBD）。CBD单体显示出β-片层夹心折叠。保守表面残基的广泛突变和胶原蛋白结合研究使我们能够鉴定蛋白质的胶原蛋白结合表面并提出可能的胶原蛋白结合模型。在每个CBD的N-末端发现十二个残基长的接头。在不存在钙的情况下，接头采用α螺旋。钙的加入使连接体解旋并将其作为新的β链锚定到三明治的远端。通过尺寸排阻色谱法和动态光散射法测定的Stokes和流体动力学半径变化证实了钙结合后接头的构象变化。该结构域在钙离子存在下变得更加刚性和有效地与胶原蛋白结合。此外，从其他梭菌物种中纯化了各种胶原酶。对它们的结构基因进行测序，以表明它们具有可变结构域组织。这意味着在基因进化过程中重复的结构域重复事件。为了揭示水解三螺旋肽底物的分子基础，我们已经产生了来自这些酶的重组催化结构域。我们开始用X射线晶体学分析它们的结构。目前，从C.溶组织I类胶原酶，其在X射线分析下。