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Drug design based on the three-dimensional structure of collagen-binding domain, and its application

基于胶原结合域三维结构的药物设计及其应用

基本信息

批准号：
12670258
负责人：
MATSUSHITA Osamu
金额：
$ 2.11万
依托单位：
Kagawa Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670258/
关键词：
Clostridium collagenase collagen-binding domain beta-sandwitch collagenous peptide

项目摘要

Tandem collagen-binding domains (CBD's) are present at the C-terminus of Clostridium histicum class I collagenase. Three-dimesional structure of the domain was determined in the presence and abscence of Ca^<2+> ion. Addition of the ion altered conformation of the N-terminal linker peptide from an alpha-helix to beta-sheets, which stabilizes the beta-sandwich domain structure and increases the substrate affinity. (Joint project with Dr. Joshua Sakon et al. University of Arkansas, U. S. A.) in order to investigate the mode of substrate binding, mutated CBD's were constructed, where various surface-oriented amino acid residues are altered. By surface plasmon resonance using a sensor chip with an immobilized collagenous peptide, G(POG)_8, we determined their binding constants against this artificial substrate. This expriment showed that a hydrophobic surface of the sandwitch plays a key role for the substrate binding.Binding of CBD against various types of collagen was studied by immunohistochemistry. Light and electron microscopic observation was performed after allowing CBD to bind to prefixed collagen-rich tissues, i.e. kidney, cartilage and aorta. CBD bound to all these tissues, but with no periodicity. CBD also bound to various types of collagen in vitro. These results suggested that CBD recognizes its triple helical confomation.We purified collagenases from three gelatinolytic Clostridia, and cloned their structural genes. Comparison of the deduced sequences revealed that they possess unique segmental structure. We could observe the dynamic rearrangements of the enzyme strucure by comparing the primary sequence of various enzymes.

串联胶原结合结构域（CBD）存在于组织梭状芽胞杆菌I类胶原酶的c端。在Ca^<2+>离子存在和缺席的情况下，确定了结构域的三维结构。离子的加入改变了n端连接肽的构象，从α -螺旋变为β -片，从而稳定了β -三明治结构域结构并增加了底物亲和力。(与Joshua Sakon博士等人的联合项目。为了研究底物结合模式，构建了突变的CBD，其中各种表面取向的氨基酸残基被改变。利用固定化胶原肽G(POG)_8传感器芯片，通过表面等离子体共振测定了它们与人工底物的结合常数。该实验表明，夹层的疏水表面对底物的结合起着关键作用。采用免疫组化方法研究CBD对不同类型胶原蛋白的结合作用。将CBD与预先固定的富含胶原的组织，即肾脏、软骨和主动脉结合后，进行光镜和电镜观察。CBD与所有这些组织结合，但没有周期性。CBD在体外也能与多种胶原蛋白结合。这些结果表明，CBD识别其三螺旋构象。我们从三个溶明胶梭菌中纯化胶原酶，并克隆了它们的结构基因。对推导出的序列进行比较，发现它们具有独特的片段结构。通过比较不同酶的一级序列，可以观察到酶结构的动态重排。