Braun Nina, Lynagh Timothy, Yu Rilei, Biggin Philip C, Pless Stephan A
Department of Drug Design and Pharmacology, University of Copenhagen , Copenhagen 2100, Denmark.
Department of Biochemistry, University of Oxford , South Parks Road, Oxford OX1 3QU, United Kingdom.
ACS Chem Neurosci. 2016 Mar 16;7(3):339-48. doi: 10.1021/acschemneuro.5b00298. Epub 2016 Jan 29.
Cys-loop receptors mediate fast synaptic transmission in the nervous system, and their dysfunction is associated with a number of diseases. While some sequence variability is essential to ensure specific recognition of a chemically diverse set of ligands, other parts of the underlying amino acid sequences show a high degree of conservation, possibly to preserve the overall structural fold across the protein family. In this study, we focus on the only two absolutely conserved residues across the Cys-loop receptor family, two Trp side chains in the WXD motif of Loop D and in the WXPD motif of Loop A. Using a combination of conventional mutagenesis, unnatural amino acid incorporation, immunohistochemistry and MD simulations, we demonstrate the crucial contributions of these two Trp residues to receptor expression and function in two prototypical Cys-loop receptors, the anion-selective GlyR α1 and the cation-selective nAChR α7. Specifically, our results rule out possible electrostatic contributions of these Trp side chains and instead suggest that the overall size and shape of this aromatic pair is required in stabilizing the Cys-loop receptor extracellular domain.
半胱氨酸环受体介导神经系统中的快速突触传递，其功能障碍与多种疾病相关。虽然一些序列变异性对于确保对化学性质多样的一系列配体的特异性识别至关重要，但基础氨基酸序列的其他部分显示出高度保守性，这可能是为了在整个蛋白质家族中保留整体结构折叠。在本研究中，我们聚焦于半胱氨酸环受体家族中仅有的两个绝对保守的残基，即环D的WXD基序和环A的WXPD基序中的两个色氨酸侧链。通过结合传统诱变、非天然氨基酸掺入、免疫组织化学和分子动力学模拟，我们证明了这两个色氨酸残基对两种典型半胱氨酸环受体（阴离子选择性甘氨酸受体α1和阳离子选择性烟碱型乙酰胆碱受体α7）的受体表达和功能的关键贡献。具体而言，我们的结果排除了这些色氨酸侧链可能的静电作用，反而表明这一芳香对的整体大小和形状对于稳定半胱氨酸环受体细胞外结构域是必需的。
