Molecular determinants of ligand discrimination in the glutamate-binding pocket of the NMDA receptor
Molecular determinants of ligand discrimination in the glutamate-binding pocket of the NMDA receptor
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DOI：
10.1016/j.neuropharm.2004.07.041
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发表时间：
2004-12-01
影响因子：
4.7
通讯作者：
Betz, H
中科院分区：
文献类型：
--
作者：
Laube, B;Schemm, R;Betz, H
谷氨酸与离子型谷氨酸受体的结合发生在由保守的S1和S2结构域构建的双叶酸结合口袋内。利用N-甲基-D-天冬氨酸((RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic)受体NR2B亚基谷氨酸结合区的晶体结构，我们通过定点突变，确定了N-甲基-D-天冬氨酸受体NR2B亚基谷氨酸结合口袋内的配体选择性和有效性的决定因素。对突变的NR2B多肽的电生理分析表明，对L-谷氨酸的亲和力有强烈的影响，但对共激动剂甘氨酸的亲和力没有影响。在19个替换中的7个替换中，我们发现完全激动剂L-谷氨酸和部分激动剂NMDA的效力存在差异。特别是，位于S1域和S2域之间的替换导致激动剂效力的改变，这表明它在转导配体结合信号中发挥了作用。竞争性拮抗剂D-AP5的抑制对取代参与稳定结合口袋闭合构象的残基高度敏感，这与拮抗剂阻止结合口袋关闭一致。此外，我们确定了预计对连接NMDA的甲基很重要的残基。总的来说，我们的数据描述了特定的侧链相互作用，这些相互作用决定了NMDA受体谷氨酸位点的配基有效性和药理作用。(C)2004爱思唯尔有限公司。保留所有权利。
Binding of glutamate to ionotropic glutamate receptors occurs within a bilobate binding pocket built from conserved S1 and S2 domains. Using the crystal structure of the binding region of the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid (AMPA)- selective GluR2 subunit, we identified determinants of ligand selectivity and efficacy within the glutamate-binding pocket of the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor by site-directed mutagenesis. Electrophysiological analyses of mutated NR2B polypeptides revealed drastic effects on the affinity Of L-glutamate but not of the co-agonist glycine. With seven out of 19 substitutions, we found differences in the potency of the full agonist L-glutamate and the partial agonist NMDA. In particular, substitutions located at the interface between the SI and S2 domains resulted in changes of agonist efficacy, suggesting a role in transducing the ligand-binding signal. Inhibition by the competitive antagonist D-AP5 was highly sensitive to replacement of residues involved in stabilization of the closed conformation of the binding pocket, consistent with antagonists preventing closure of the binding pocket. In addition, we identified residues predicted to be important for liganding the methyl group of NMDA. Collectively our data describe specific side chain interactions that determine ligand efficacy and pharmacology at the glutamate site of the NMDA receptor. (C) 2004 Elsevier Ltd. All rights reserved.