Identification of residues important for ligand binding of thromboxane A2 receptor in the second extracellular loop using the NMR experiment-guided mutagenesis approach
Identification of residues important for ligand binding of thromboxane A2 receptor in the second extracellular loop using the NMR experiment-guided mutagenesis approach
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DOI：
10.1074/jbc.m209337200
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发表时间：
2003-03-28
影响因子：
4.8
通讯作者：
Ruan, KH
中科院分区：
文献类型：
--
作者：
So, SP;Wu, JX;Ruan, KH
血栓素 A(2) 受体 (TP) 的第二个细胞外环 (eLP2) 被认为参与配体结合。通过二维 H-1 NMR 实验，我们组确定了模拟 eLP2 的约束合成肽的整体三维结构（Ruan, K: H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001) Biochemistry 40, 275-280）。为了进一步鉴定参与配体结合的残基，使用 TP 受体拮抗剂 SQ29,548 与合成肽相互作用。分别对肽SQ29,548和肽SQ29,548进行高分辨率二维H-1 NMR实验NOESY和TOCSY。通过完成的 H-1 NMR 归属和比较不同的光谱，在具有 SQ29,548 的肽的 NOESY 光谱上观察到额外的峰，这表明 eLP2 的 Val(176)、Leu(185)、Thr(186) 和 Leu(187) 残基与 SQ29,548 的 H2、H7 和 H8 位置处的残基接触。使用定点诱变来确认天然人TP受体上可能的配体结合位点。四个残基中的每一个都突变为同一组中具有不同结构或不同电荷的残基。然后测试突变受体的配体结合活性。具有V176L突变体的受体保留了与SQ29,548的结合活性。所有其他突变均导致与 SQ29,548 的结合活性降低或丧失。这些诱变结果支持 NMR 实验的预测，其中 Val(176)、Leu(185)、Thr(186) 和 Leu(187) 是参与配体结合的可能残基。这些信息有助于理解血栓素A(2)与重要受体结合的分子机制及其信号转导。
The second extracellular loop (eLP2) of the thromboxane A(2) receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional H-1 NMR experiments, the overall three-dimensional structure of a constrained synthetic peptide mimicking the eLP2 had been determined by our group (Ruan, K: H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001) Biochemistry 40, 275-280). To further identify the residues involved in ligand binding, a TP receptor antagonist, SQ29,548 was used to interact with the synthetic peptide. High resolution two-dimensional H-1 NMR experiments, NOESY, and TOCSY were performed for the peptide, SQ29,548, and peptide with SQ29,548, respectively. Through completed H-1 NMR assignment and by comparing the different spectra, extra peaks were observed on the NOESY spectrum of the peptide with SQ29,548, which implied the contacts between residues of eLP2 at Val(176), Leu(185), Thr(186), and Leu(187) with SQ29,548 at position H2, H7, and H8. Site-directed mutagenesis was used to confirm the possible ligand-binding sites on native human TP receptor. Each of the four residues was mutated to the residues either in the same group, with different structure or different charged. The mutated receptors were then tested for their ligand binding activity. The receptor with V176L mutant retained binding activity to SQ29,548. All other mutations resulted in decreased or lost binding activity to SQ29,548. These mutagenesis results supported the prediction from NMR experiments in which Val(176), Leu(185), Thr(186), and Leu(187) are the possible residues involved in ligand binding. This information facilitates the understanding of the molecular mechanism of thromboxane A(2) binding to the important receptor and its signal transduction.