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

串联胶原结合结构域（CBD）存在于组织梭菌I类胶原酶的C末端。在Ca^<2+>离子存在和掺杂的条件下，测定了该畴的三维结构。离子的添加改变了N-末端接头肽的构象，从α-螺旋变为β-折叠，这稳定了β-夹心结构域结构并增加了底物亲和力。（与约书亚萨孔博士等的联合项目，美国阿肯色州大学。S. A.）为了研究底物结合的模式，构建了突变的CBD，其中各种表面取向的氨基酸残基被改变。利用表面等离子体共振技术，用固定化胶原肽G（POG）_8的传感器芯片，测定了它们与人工底物的结合常数。实验结果表明，CBD的疏水表面对底物的结合起着关键作用。在允许CBD结合到预先固定的富含胶原的组织（即肾脏、软骨和主动脉）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在体外也与各种类型的胶原蛋白结合。我们从三种溶胶梭菌中纯化胶原酶，并克隆了它们的结构基因。比较推导的序列，发现它们具有独特的节段结构。通过比较各种酶的一级序列，可以观察到酶结构的动态重排。

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.