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

生物反应由活性维生素D调节至基因表达水平，由维生素D受体（VDR）绑定-配体复合体与目标基因。这种基因转移中的一个关键因素是VDR依赖于该结构的三维结构。在这个研究项目中，直接研究维生素D的构象与VDR结合，我们提议使用i-D119 i-D1 F-NMR与荧光维生素D模拟物作为探测化合物。在添加中，我们调查维生素D-VDR使用荧光标记的维生素D模拟的内部交互。低温度D 119-D-1F-NMR Spectra of Successfully synthesized 4，4-difluoro-and 19-fluorovitamin D模拟显示出分离的信号叠加到两个A-环构象和那些模拟显示出与高亲和力结合的VDR（Kd= ca.）10.D1 -9） indicating to be useful probe to analyze the VDR-bound A-ring conformation of维生素D. VDR蛋白质被表达出来了。 ... More E. E.大肠杆菌BL 21（DE 3）与pET-14 b等离子体载体转染，含有rat cDNA插入。维生素D服用的两把椅子形成的A环，即α-和β-形式。D119-D1 F-NMR光谱的VDR-19-荧光素D复合体展示了A环采用的椅子α-构象。最近，活性维生素D-突变VDR复合体的X射线晶体结构被确定并在复合体中，自然配体和使用β-构象。需要进行更多详细的调查，以确定与生物活性表达相关的A环构象的活性形式。我们设计并成功地合成了两项新颖的荧光标签维生素D模拟。由于VDR与自然配体的关联性较小（0.1%），因此VDR绑定形式被估计为仍然具有排他性（B/F ： ca）。10イD18エD1） on the basis of Kd of the natural ligand。对整个细胞配体相互作用的视觉观测正在通过荧光模拟器的使用进行当前的进展。Less（低）

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