Structural Dynamics of the Glycine-binding Domain of the N-Methyl-D-Aspartate Receptor
Structural Dynamics of the Glycine-binding Domain of the N-Methyl-D-Aspartate Receptor
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DOI：
10.1074/jbc.m114.605436
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发表时间：
2015-01-09
影响因子：
4.8
通讯作者：
Jayaraman, Vasanthi
中科院分区：
文献类型：
--
作者：
Dolino, Drew M.;Cooper, David;Jayaraman, Vasanthi
N-甲基-D-天冬氨酸受体介导中枢神经系统中兴奋性神经传递的慢成分。这些受体是含有甘氨酸和谷氨酸结合亚基的专性异聚体。配体与受体的双叶激动剂结合结构域结合。以前的X-射线结构的甘氨酸结合结构域的NMDA受体之间的部分和全部激动剂结合的结构没有显着的变化。在这里，我们已经使用单分子荧光共振能量转移（smFRET）的裂缝关闭构象状态，受体的甘氨酸结合结构域的拮抗剂5，7-二氯犬尿烯酸（DCKA），部分激动剂1-氨基-1-环丁烷羧酸（ACBC）和L-丙氨酸，和完全激动剂甘氨酸和D-丝氨酸的存在下采取。对于这些研究，我们已经将非天然氨基酸对乙酰基-L-苯丙氨酸与荧光团的酰肼衍生物的蛋白质的特异性标记。单分子荧光共振能量转移数据表明，激动剂结合结构域可以采用宽范围的裂缝闭合状态，在不同功效的配体所占据的状态中具有显著重叠。不同之处在于蛋白质的分数在一个更封闭的裂缝的形式，与完全激动剂具有更大的分数在封闭的裂缝的形式，这表明配体的能力，选择这些国家可以决定的程度激活。
N-Methyl-D-aspartate receptors mediate the slow component of excitatory neurotransmission in the central nervous system. These receptors are obligate heteromers containing glycine- and glutamate-binding subunits. The ligands bind to a bilobed agonist-binding domain of the receptor. Previous x-ray structures of the glycine-binding domain of NMDA receptors showed no significant changes between the partial and full agonist-bound structures. Here we have used single molecule fluorescence resonance energy transfer (smFRET) to investigate the cleft closure conformational states that the glycine-binding domain of the receptor adopts in the presence of the antagonist 5,7-dichlorokynurenic acid (DCKA), the partial agonists 1-amino-1-cyclobutanecarboxylic acid (ACBC) and L-alanine, and full agonists glycine and D-serine. For these studies, we have incorporated the unnatural amino acid p-acetyl-L-phenylalanine for specific labeling of the protein with hydrazide derivatives of fluorophores. The single molecule fluorescence resonance energy transfer data show that the agonist-binding domain can adopt a wide range of cleft closure states with significant overlap in the states occupied by ligands of varying efficacy. The difference lies in the fraction of the protein in a more closed-cleft form, with full agonists having a larger fraction in the closed-cleft form, suggesting that the ability of ligands to select for these states could dictate the extent of activation.