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Three-dimensional structure of vitamin D ligand-receptor complex and its intracellular interaction

维生素D配体-受体复合物的三维结构及其细胞内相互作用

基本信息

批准号：
10671985
负责人：
SHIMIZU Masato
金额：
$ 1.22万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Biological responses mediated by the active vitamin D are regulated at the level of gene expression by binding of the vitamin D receptor (VDR)-ligand complex to the target gene. The key factor in this gene transactivation is the three-dimensional structure of VDR dependent on the ligand conformation. In this research project, to directly examine the vitamin D conformation binding to the VDR, we proposed the use of ィイD119ィエD1F-NMR with fluorinated vitamin D analogs as probe compounds. In addition, we investigate the intracellular interaction of vitamin D-VDR using fluorescent-labeled vitamin D analogs.The low temperature ィイD119ィエD1F-NMR spectra of successfully synthesized 4,4-difluoro- and 19-fluorovitamin D analogs showed separated signals ascribed to two-A-ring conformations and those analogs were shown to bind to the VDR with high affinity (Kd= ca. 10ィイD1-9ィエD1M) indicating to be useful probe to analyze the VDR-bound A-ring conformation of vitamin D. The VDR protein was expressed in … More E. coli BL21(DE3) transfected with pET-14b plasmid vector containing rat cDNA insert. The A-ring of vitamin D adopts two chair conformations, the α- and β-form. The ィイD119ィエD1F-NMRspectrum of VDR-19-fluorovitamin D complex showed that the A-ring adopts a chair α-conformation. Recently, X-ray crystal structure of the active vitamin D-mutant VDR complex was determined and in the complex, the natural ligand occupies β-conformation . More detail investigations are required to determine the active form of the A-ring conformation associated with the expression of biological activities.We designed and successfully synthesized two novel fluorescent-labeled vitamin D analogs. Though the binding affinity of those analogs for VDR was small (0.1%) relative to the natural ligand, the VDR-bound form is estimated to be still exclusive (B/F : ca. 10ィイD18ィエD1) on the basis of Kd of the natural ligand. Visual observation of the whole cell-ligand interaction is currently progressing through the use of the fluorescent analogs. Less

由活性维生素D介导的生物反应通过维生素D受体（VDR）-配体复合物与靶基因的结合在基因表达水平上进行调节。该基因反式激活的关键因素是VDR的三维结构依赖于配体构象。在本研究项目中，为了直接检测维生素D与VDR的构象结合，我们提出了使用氟化维生素D类似物作为探针化合物的D1 F-NMR。此外，我们还利用荧光标记的维生素D类似物研究了维生素D-VDR在细胞内的相互作用。成功合成的4，4-二氟和19-氟维生素D类似物的低温核磁共振D119核磁共振D1 F-NMR谱显示出归因于两个A环构象的分离信号，并且这些类似物显示出与VDR的高亲和力结合（Kd=约1.5）。10 kDa D1-9 kDa D1 M），是分析维生素D的VDR结合A环构象的有用探针。VDR蛋白在大肠杆菌中表达。 ...更多信息 E.将含有大鼠cDNA插入片段的pET-14 b质粒载体转染到大肠杆菌BL 21（DE 3）中。维生素D的A环具有两种椅式构象，α-和β-形式。VDR-19-氟维生素D复合物的D119-D1 F-NMR谱表明，其A环为椅式α构象。最近，活性维生素D-突变型VDR复合物的X-射线晶体结构被测定，在复合物中，天然配体占据β-构象。我们设计并成功合成了两种新型的荧光标记维生素D类似物。尽管这些类似物对VDR的结合亲和力相对于天然配体较小（0.1%），但估计VDR结合形式仍是排他性的（B/F：约100%）。10 μ g D18 μ g D1）。整个细胞-配体相互作用的视觉观察目前正在通过使用荧光类似物进行。少