Solution NMR of acetylcholine binding protein reveals agonist-mediated conformational change of the C-loop
Solution NMR of acetylcholine binding protein reveals agonist-mediated conformational change of the C-loop
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DOI：
10.1124/mol.106.027185
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发表时间：
2006-10-01
影响因子：
3.6
通讯作者：
Sine, Steven M.
中科院分区：
文献类型：
--
作者：
Gao, Fan;Mer, Georges;Sine, Steven M.
先前的X射线结晶学、分子动力学模拟、荧光光谱和乙酰胆碱结合蛋白(AChBP)的氢-氢交换表明，当激动剂与激动剂结合后，结合部位的C环向内拉，覆盖激动剂。在本研究中，我们使用高分辨率溶液核磁共振来监测没有和有乙酰胆碱(ACh)结合的C-环的化学环境的变化。用[N-15]半胱氨酸取代天然半胱氨酸123、136、187和188，分别提供了半胱氨酸和C-环化学环境的内在监测器。Apo-AChBP的二维横向弛豫优化光谱N-15-H-1HSQC谱显示，半胱氨酸基有七个分辨良好的交叉峰。用丝氨酸取代半胱氨酸187和188的AChBP的光谱只显示了两个主要的串音，对应于来自半胱氨酸环的半胱氨酸123和136，使共振归属成为可能。ACh结合后，与C环半胱氨酸相关的五个交叉峰缩合成两个在载脂蛋白光谱中没有观察到的交叉峰，表明C环的构象范围有限，化学环境发生了变化。结果表明，同位素半胱氨酸可以被掺入真核来源表达的AChBP的特定位置，C-环在没有ACh的情况下呈现多种构象，但其构象受到ACh结合的限制。这些集体发现提示了AChBP和相关的Cys-loop受体中激动剂识别的结构机制。
Previous X-ray crystallography, molecular dynamics simulation, fluorescence spectroscopy, and deuterium-hydrogen exchange of acetylcholine binding protein (AChBP) suggest that after binding of the agonist, the C-loop at the periphery of the binding site draws inward to cap the site and envelop the agonist. In this study, we use high-resolution solution NMR to monitor changes in the chemical environment of the C-loop without and with acetylcholine (ACh) bound. Substitution of [N-15] cysteine for the native cysteines 123, 136, 187, and 188 provided intrinsic monitors of the chemical environments of the Cys-and C-loops, respectively. Two-dimensional transverse relaxation-optimized spectroscopy N-15-H-1 HSQC spectroscopy of apo-AChBP revealed seven well resolved cross-peaks for the group of cysteines. The spectrum of AChBP with Ser substituted for Cys 187 and 188 shows only two main crosspeaks, corresponding to Cys 123 and 136 from the Cys-loop, enabling resonance assignments. After binding of ACh, the five cross-peaks associated with cysteines from the C-loop condense into two predominant cross-peaks not observed in the spectrum from the apo protein, indicating a restricted range of conformations and change in chemical environment of the C-loop. The results show that isotopic cysteine can be incorporated into specified positions of AChBP expressed from a eukaryotic source, that the C-loop assumes multiple conformations without ACh, but that its conformation becomes restricted with ACh bound. The collective findings suggest a structural mechanism for agonist recognition in AChBP and related Cys-loop receptors.