Armstrong Neali, Mayer Mark, Gouaux Eric
Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA.
Proc Natl Acad Sci U S A. 2003 May 13;100(10):5736-41. doi: 10.1073/pnas.1037393100. Epub 2003 May 2.
The (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) receptor discriminates between agonists in terms of binding and channel gating; AMPA is a high-affinity full agonist, whereas kainate is a low-affinity partial agonist. Although there is extensive literature on the functional characterization of partial agonist activity in ion channels, structure-based mechanisms are scarce. Here we investigate the role of Leu-650, a binding cleft residue conserved among AMPA receptors, in maintaining agonist specificity and regulating agonist binding and channel gating by using physiological, x-ray crystallographic, and biochemical techniques. Changing Leu-650 to Thr yields a receptor that responds more potently and efficaciously to kainate and less potently and efficaciously to AMPA relative to the WT receptor. Crystal structures of the Leu-650 to Thr mutant reveal an increase in domain closure in the kainate-bound state and a partially closed and a fully closed conformation in the AMPA-bound form. Our results indicate that agonists can induce a range of conformations in the GluR2 ligand-binding core and that domain closure is directly correlated to channel activation. The partially closed, AMPA-bound conformation of the L650T mutant likely captures the structure of an agonist-bound, inactive state of the receptor. Together with previously solved structures, we have determined a mechanism of agonist binding and subsequent conformational rearrangements.
（S）-2-氨基-3-（3-羟基-5-甲基-4-异恶唑）丙酸（AMPA）受体在结合和通道门控方面能够区分激动剂；AMPA是一种高亲和力的完全激动剂，而海人酸是一种低亲和力的部分激动剂。尽管关于离子通道中部分激动剂活性的功能特性已有大量文献，但基于结构的机制却很少见。在这里，我们通过生理、X射线晶体学和生化技术，研究了AMPA受体中保守的结合裂隙残基Leu-650在维持激动剂特异性以及调节激动剂结合和通道门控方面的作用。将Leu-650替换为Thr后产生的受体，相对于野生型受体，对海人酸的反应更有效且更强烈，而对AMPA的反应则较弱且效果较差。Leu-650突变为Thr的晶体结构显示，在结合海人酸的状态下结构域闭合增加，在结合AMPA的形式下呈现部分闭合和完全闭合的构象。我们的结果表明，激动剂可在GluR2配体结合核心诱导一系列构象，且结构域闭合与通道激活直接相关。L650T突变体结合AMPA时的部分闭合构象可能捕获了受体与激动剂结合的非活性状态的结构。结合之前解析的结构，我们确定了激动剂结合及随后构象重排的机制。
