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）存在于梭状芽胞杆菌I类胶原酶的C末端。在存在和吸收Ca^<2+>离子的情况下确定了域的三个时光结构。 N端连接器肽从α-螺旋到Beta片的添加改变了N末端接头肽，从而稳定β-Sandwich域结构并增加了底物亲和力。（与美国阿肯色大学的Joshua Sakon等人的联合项目）为了研究底物结合的模式，构建了突变的CBD，其中改变了各种面向表面的氨基酸残基。通过表面等离子体的共振，使用传感器芯片具有固定的胶原肽G（POG）_8，我们确定了它们与该人工基材的结合常数。这种拔出表明，三核的疏水表面对底物结合起着关键作用。通过免疫组织化学研究了CBD对各种类型的胶原蛋白的结合。在允许CBD结合前缀富含胶原蛋白的组织，即肾脏，软骨和主动脉之后，进行了光和电子显微镜观察。 CBD与所有这些组织结合，但没有周期性。 CBD还与各种类型的体外结合。这些结果表明，CBD认识到其三重螺旋抗性。我们从三个明胶裂解梭菌中纯化了胶原酶，并克隆了其结构基因。推论序列的比较表明它们具有独特的节段结构。我们可以通过比较各种酶的一级序列来观察酶构型的动态重排。