Structural analysis of yoked chorionic gonadotropin-luteinizing hormone receptor ectodomain complexes by circular dichroic spectroscopy
Structural analysis of yoked chorionic gonadotropin-luteinizing hormone receptor ectodomain complexes by circular dichroic spectroscopy
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DOI：
10.1210/me.2002-0349
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发表时间：
2003-07-01
影响因子：
--
通讯作者：
Puett, D
中科院分区：
文献类型：
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作者：
Fralish, GB;Dattilo, B;Puett, D
异二聚体糖蛋白激素绒毛膜促性腺激素（CG）与七螺旋LH受体N-末端胞外域（ECD）结合，其中大部分已被建模为富含亮氨酸的重复蛋白。在这项研究中，我们表达并纯化了三个单链N-CG-ECD-C复合物，一个包含全长ECD，1-341（由外显子1-10和11的一部分编码），和两个C-末端ECD缺失片段，1-294（由外显子1-10编码）和1-180（由外显子1-7编码）。通过Western印迹分析和圆二色性（CD）对融合蛋白（包括偶联CG（N-beta-alpha-C））进行表征。对CG-ECD融合蛋白获得的CD光谱和减去CG贡献后的每种差异光谱的分析，产生了与同源性模型中预测的重复β链/α螺旋折叠一致的二级结构。当从ECD中除去C-末端47个氨基酸残基时，观察到螺旋度显著降低。去除额外的114个残基，即由外显子8-10编码的区域，导致较少的螺旋残基的损失。这些结果表明，铰链区的ECD，预测仅包含有限的二级结构，相互作用，并稳定的配体占据的N-末端部分。此外，结果支持重复折叠，与LHR ECD的拟议模型一致。
Binding of the heterodimeric glycoprotein hormone, chorionic gonadotropin (CG), occurs to the heptahelical LH receptor N-terminal ectodomain (ECD), a large portion of which has been modeled as a leucine-rich repeat protein. In this study, we expressed and purified three single chain N-CG-ECD-C complexes, one comprising the full-length ECD, 1-341 (encoded by exons 1-10 and a portion of 11), and two C-terminal ECD deletion fragments, 1-294 (encoded by exons 1-10) and 1-180 (encoded by exons 1-7). The fusion proteins, including yoked CG (N-beta-alpha-C), were characterized by Western blot analysis and circular dichroism (CD). Analysis of the CD spectra obtained on the CG-ECD fusion proteins, and of the difference spectrum of each after subtracting the CG contribution, yielded secondary structures consistent with a repeating beta-strand/alpha-helix fold as predicted in the homology model. A marked decrease in helicity was observed when the C-terminal 47 amino acid residues were removed from the ECD. Removal of an additional 114 residues, i.e. the region encoded by exons 8-10, results in the loss of fewer helical residues. These results suggest that the hinge region of the ECD, predicted to contain only limited secondary structure, interacts with and stabilizes the ligand-occupied N-terminal portion. Furthermore, the results support a repeating fold, consistent with the proposed model for the LHR ECD.