Mendieta Jesús, Gago Federico, Ramírez Galo
Centro de Biología Molecular Severo Ochoa, Universidad Autónoma, E-28049 Cantoblanco, Madrid, Spain.
Biochemistry. 2005 Nov 8;44(44):14470-6. doi: 10.1021/bi051084x.
Guanine nucleotides behave as competitive antagonists at ionotropic glutamate receptors and show neuroprotective activity in different experimental excitotoxicity paradigms, both in vivo and in cultured cell preparations. Taking 5'-GMP as the reference nucleotide, we have tried to understand how these molecules interact with the agonist-binding site of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor. Using a crystallographic model of the ligand-binding core of the GluR2 receptor in complex with kainate, we have previously analyzed the structural changes associated to the binding of agonists to the receptor and suggested a mechanism for the coupling of agonist binding to channel gating. In the present investigation we used the structure of the apo form of the receptor to probe the primary interactions between GMP and GluR2 by means of an automated docking program. A targeted molecular dynamics (TMD) simulation procedure was subsequently used to force the closing of the protein and to study the rearrangement of the ligand and surrounding amino acids. The resulting structure provides a plausible model of the nucleotide-receptor complex. Indirect support for the validity of our approach was obtained when the same methodology was shown to yield structures of the kainate-GluR2 and 6,7-dinitroquinoxaline-2,3-dione (DNQX)-GluR2 complexes that were in very good agreement with the published crystallographic structures. Both the stacking interaction between the phenyl ring of Tyr73 and the purine ring of GMP and a salt bridge between the phosphate group of GMP and Arg108 in the S1 domain, together with several hydrogen bonds, are proposed to secure the anchoring of GMP to the agonist-binding site. Unlike conventional competitive antagonists, such as DNQX, occupancy of the site by GMP still allows receptor segments S1 and S2 to close tightly around GMP without interacting with the critical residue Glu209 that triggers channel opening. Thus, GMP appears to be rather a false agonist than a competitive antagonist. This fact and the nature of the energy barriers that stabilize GMP bound to the closed form of the receptor provide an explanation for the unusual behavior of some guanine nucleotides in ligand-displacement experiments.
鸟嘌呤核苷酸在离子型谷氨酸受体上表现为竞争性拮抗剂，并且在体内和培养细胞制剂的不同实验性兴奋性毒性范式中均显示出神经保护活性。以5'-鸟苷酸（5'-GMP）作为参考核苷酸，我们试图了解这些分子如何与红藻氨酸盐（AMPA）受体GluR2的激动剂结合位点相互作用。利用与红藻氨酸盐复合的GluR2受体配体结合核心的晶体学模型，我们之前分析了与激动剂结合到受体相关的结构变化，并提出了激动剂结合与通道门控偶联的机制。在本研究中，我们使用受体的无配体形式的结构，通过自动对接程序探究GMP与GluR2之间的主要相互作用。随后使用靶向分子动力学（TMD）模拟程序促使蛋白质关闭，并研究配体和周围氨基酸的重排。所得结构提供了核苷酸-受体复合物的合理模型。当相同方法显示产生的红藻氨酸盐-GluR2和6,7-二硝基喹喔啉-2,3-二酮（DNQX）-GluR2复合物的结构与已发表的晶体学结构非常吻合时，我们的方法的有效性得到了间接支持。酪氨酸73的苯环与GMP的嘌呤环之间的堆积相互作用以及S1结构域中GMP的磷酸基团与精氨酸108之间的盐桥，连同几个氢键，被认为确保了GMP锚定到激动剂结合位点。与传统的竞争性拮抗剂（如DNQX）不同，GMP占据该位点仍允许受体片段S1和S2紧密围绕GMP关闭，而不与触发通道开放的关键残基谷氨酸209相互作用。因此，GMP似乎更像是一种假激动剂而非竞争性拮抗剂。这一事实以及稳定与受体封闭形式结合的GMP的能量屏障的性质，为一些鸟嘌呤核苷酸在配体置换实验中的异常行为提供了解释。
