Dolino Drew M, Cooper David, Ramaswamy Swarna, Jaurich Henriette, Landes Christy F, Jayaraman Vasanthi
From the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, Texas 77030 and.
the Departments of Chemistry and.
J Biol Chem. 2015 Jan 9;290(2):797-804. doi: 10.1074/jbc.M114.605436. Epub 2014 Nov 17.
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.
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。这些受体是包含甘氨酸结合亚基和谷氨酸结合亚基的 obligate 异源二聚体。配体与受体的双叶激动剂结合结构域结合。之前 NMDA 受体甘氨酸结合结构域的 X 射线结构显示，部分激动剂结合结构和完全激动剂结合结构之间没有显著变化。在这里，我们使用单分子荧光共振能量转移（smFRET）来研究受体的甘氨酸结合结构域在拮抗剂 5,7-二氯犬尿氨酸（DCKA）、部分激动剂 1-氨基-1-环丁烷羧酸（ACBC）和 L-丙氨酸以及完全激动剂甘氨酸和 D-丝氨酸存在下所采用的裂隙闭合构象状态。对于这些研究，我们引入了非天然氨基酸对乙酰-L-苯丙氨酸，以便用荧光团的酰肼衍生物对蛋白质进行特异性标记。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多种裂隙闭合状态，不同效能的配体所占据的状态有显著重叠。区别在于处于更闭合裂隙形式的蛋白质比例，完全激动剂在闭合裂隙形式中的比例更大，这表明配体选择这些状态的能力可能决定激活程度。
