Department of Biochemistry, Cambridge, UK.
Biophys J. 2012 Nov 21;103(10):2071-81. doi: 10.1016/j.bpj.2012.10.016. Epub 2012 Nov 20.
RDL receptors are GABA-activated inhibitory Cys-loop receptors found throughout the insect CNS. They are a key target for insecticides. Here, we characterize the GABA binding site in RDL receptors using computational and electrophysiological techniques. A homology model of the extracellular domain of RDL was generated and GABA docked into the binding site. Molecular dynamics simulations predicted critical GABA binding interactions with aromatic residues F206, Y254, and Y109 and hydrophilic residues E204, S176, R111, R166, S176, and T251. These residues were mutated, expressed in Xenopus oocytes, and their functions assessed using electrophysiology. The data support the binding mechanism provided by the simulations, which predict that GABA forms many interactions with binding site residues, the most significant of which are cation-π interactions with F206 and Y254, H-bonds with E204, S205, R111, S176, T251, and ionic interactions with R111 and E204. These findings clarify the roles of a range of residues in binding GABA in the RDL receptor, and also show that molecular dynamics simulations are a useful tool to identify specific interactions in Cys-loop receptors.
RDL 受体是 GABA 激活的抑制性 Cys 环受体，存在于昆虫中枢神经系统的各个部位。它们是杀虫剂的重要靶标。在这里，我们使用计算和电生理技术来描述 RDL 受体中的 GABA 结合位点。生成了 RDL 细胞外结构域的同源模型，并将 GABA 对接至结合位点。分子动力学模拟预测了 GABA 与芳香族残基 F206、Y254 和 Y109 以及亲水性残基 E204、S176、R111、R166、S176 和 T251 之间的关键 GABA 结合相互作用。对这些残基进行了突变，在非洲爪蟾卵母细胞中表达，并用电生理学评估它们的功能。数据支持模拟提供的结合机制，该机制预测 GABA 与结合位点残基形成许多相互作用，其中最重要的是与 F206 和 Y254 的阳离子-π 相互作用、与 E204、S205、R111、S176、T251 的氢键以及与 R111 和 E204 的离子相互作用。这些发现阐明了 RDL 受体中 GABA 结合的一系列残基的作用，还表明分子动力学模拟是识别 Cys 环受体中特定相互作用的有用工具。
