摘要
目的 研究VLDL R中 8个配体结合重复序列 (ligand bindingrepeats ,LBR)在配体结合中的作用并探讨结合位点的结构。方法 采用基因缺失诱变方法 ,构建不同LBR缺失的VLDL R重组体。将其分别导入无LDL R功能性表达的ldl A7细胞。配体结合实验观察转染细胞与荧光标记的VLDL、β VLDL的结合能力 ,并采用同源建模的方法预测受体N 端 32 8个氨基酸的配体结合域的空间结构。结果 配体结合实验显示LBR1和LBR2对受体结合VLDL、β VLDL最为重要 ;LBR3对结合VLDL也有重要作用 ,但对结合 β VLDL无明显影响。模型显示受体N 端32 8个氨基酸的配体结合域呈现弧形口袋样结构 ,前 3个LBR结构紧凑 ,呈棒状 ,负电荷相对集中 ,与功能特性吻合 ,LBR5与LBR6之间的连接区赋予口袋结构一定的伸缩性 ,适应配体大小的变化。结论 受体N 端的前 3个LBR含有与配体结合的位点。该区域特定的空间结构与理化特性是结合功能的重要基础。
Objective To study the contribution of VLDL-R's 8 repeats to ligand-binding and to explore the structure of ligand-binding sites.Methods By using method of oligonucleotide-directed mutagenesis, VLDL-R recombinants lacking different repeats were constructed and transfected into ldl-A7 cells. The ligand binding assay was conducted with DiI labeled VLDL and β-VLDL on the transfected cells. The method of protein structure prediction was adapted to analyze the 3D structure of the 328 amino acids sequence at the receptor's N-end. Results Ligand binding assay showed that repeat 1 and repeat 2 were the most important LBRs in binding with VLDL and β-VLDL; Repeat 3 also important for binding VLDL. LBR1-8 showed an arc pocket-like structure with a tightly clavate region clustered with negative charge formed by the front 3 LBRs. A linker between LBR 5 and LBR6 conferred the structure relative flexibility. Conclusion The front 3 LBRs in N-end contain ligand binding sites. Special arc pocket-like structure is the bases of the receptor's binding function.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2004年第5期526-530,共5页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目 (No 396 70 16 2 )
