GABA与五聚体配体门控离子通道结合的自由能景观及其突变破坏
The free energy landscape of GABA binding to a pentameric ligand-gated ion channel and its disruption by mutations
摘要：
Cys环超家族的五聚体配体门控离子通道（pLGIC）是介导快速突触传递的重要神经受体。它们被神经递质的结合激活，但这个过程的细节仍然没有完全了解。作为一个典型的pLGIC，在这里，我们选择了昆虫对狄氏剂的抗性（RDL）受体，参与对杀虫剂的抗性，并在原子水平上研究神经递质GABA与其胞外结构域的结合。我们实现了这一点，通过$μ$秒漏斗metadaptics模拟，有效地提高了采样的束缚和未束缚状态，通过使用漏斗形的限制潜力，以限制在溶剂中的探索。我们揭示了结合过程中的事件序列，从溶剂中的GABA的捕获到其在结合口袋中的带电残基Arg111和Glu204之间的钉扎。我们表征了相关的自由能景观在野生型RDL受体和两种突变形式，其中关键残基Arg111和Glu204突变为Ala。实验上，这些突变产生非功能性通道，这反映在由于基本相互作用的丧失而降低的配体结合亲和力中。我们还分析了关键环C的动力学行为，其开放允许GABA进入结合位点，而其关闭将配体锁定到蛋白质中。RDL受体与其他pLGIC共享结构和功能特征，因此我们的工作概述了一个有价值的协议，以研究配体与pLGIC的结合，超越传统的对接和分子动力学技术。
英文摘要：
Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop superfamily are important neuroreceptors that mediate fast synaptic transmission. They are activated by the binding of a neurotransmitter, but the details of this process are still not fully understood. As a prototypical pLGIC, here we choose the insect resistance to dieldrin (RDL) receptor, involved in the resistance to insecticides, and investigate the binding of the neurotransmitter GABA to its extracellular domain at the atomistic level. We achieve this by means of $μ$-sec funnel-metadynamics simulations, which efficiently enhance the sampling of bound and unbound states by using a funnel-shaped restraining potential to limit the exploration in the solvent. We reveal the sequence of events in the binding process, from the capture of GABA from the solvent to its pinning between the charged residues Arg111 and Glu204 in the binding pocket. We characterize the associated free energy landscapes in the wild-type RDL receptor and in two mutant forms, where the key residues Arg111 and Glu204 are mutated to Ala. Experimentally these mutations produce non-functional channels, which is reflected in the reduced ligand binding affinities, due to the loss of essential interactions. We also analyze the dynamical behaviour of the crucial loop C, whose opening allows the access of GABA to the binding site, while its closure locks the ligand into the protein. The RDL receptor shares structural and functional features with other pLGICs, hence our work outlines a valuable protocol to study the binding of ligands to pLGICs beyond conventional docking and molecular dynamics techniques.
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