Department of Pharmacology, National University of Singapore, Singapore.
Mol Brain. 2008 Nov 18;1:16. doi: 10.1186/1756-6606-1-16.
Phenylethanolamines selectively bind to NR2B subunit-containing N-methyl-D-aspartate-subtype of ionotropic glutamate receptors and negatively modulate receptor activity. To investigate the structural and functional properties of the ifenprodil binding domain on the NR2B protein, we have purified a soluble recombinant rat NR2B protein fragment comprising the first ~400 amino acid amino-terminal domain (ATD2B) expressed in E. coli. Spectral measurements on refolded ATD2B protein demonstrated specific binding to ifenprodil. We have used site-directed mutagenesis, circular dichroism spectroscopy and molecular modeling to obtain structural information on the interactions between critical amino acid residues and ifenprodil of our soluble refolded ATD2B proteins. Ligand-induced changes in protein structure were inferred from changes in the circular dichroism spectrum, and the concentration dependence of these changes was used to determine binding constants for ifenprodil and its analogues.
Ligand binding of ifenprodil, RO25,6981 and haloperidol on soluble recombinant ATD2B determined from circular dichroism spectroscopy yielded low-to-high micromolar equilibrium constants which concurred with functional IC₅₀ measurement determined in heterologously expressed NR1/NR2B receptors in Xenopus oocytes. Amino acid residue substitutions of Asp101, Ile150 and Phe176 with alanine residue within the ATD2B protein altered the recombinant protein dissociation constants for ifenprodil, mirroring the pattern of their functional phenotypes. Molecular modeling of ATD2B as a clam-shell-like structure places these critical residues near a putative ligand binding site.
We report for the first time biochemical measurements show that the functional measurements actually reflect binding to the ATD of NR2B subunit. Insights gained from this study help advance the theory that ifenprodil is a ligand for the ATD of NR2B subunit.
苯乙醇胺选择性地与包含 NMDA 型离子型谷氨酸受体 NR2B 亚基的受体结合，并负调节受体活性。为了研究ifenprodil 在 NR2B 蛋白上的结合域的结构和功能特性，我们在大肠杆菌中表达了一种可溶性重组大鼠 NR2B 蛋白片段，该片段包含第一个~400 个氨基酸的氨基末端结构域（ATD2B）。对重折叠 ATD2B 蛋白的光谱测量表明其与 ifenprodil 特异性结合。我们使用定点突变、圆二色性光谱和分子建模来获得我们可溶性重折叠 ATD2B 蛋白中关键氨基酸残基与 ifenprodil 相互作用的结构信息。从圆二色性光谱的变化推断出配体诱导的蛋白质结构变化，并使用这些变化的浓度依赖性来确定 ifenprodil 及其类似物的结合常数。
从圆二色性光谱测定的可溶性重组 ATD2B 上 ifenprodil、RO25、6981 和氟哌啶醇的配体结合，得出低至高微摩尔平衡常数，与在非洲爪蟾卵母细胞中异源表达的 NR1/NR2B 受体中功能性 IC₅₀ 测量结果一致。在 ATD2B 蛋白中用丙氨酸残基取代 Asp101、Ile150 和 Phe176 氨基酸残基改变了重组蛋白对 ifenprodil 的解离常数，反映了其功能表型的模式。将 ATD2B 作为蛤壳样结构的分子建模将这些关键残基置于一个假定的配体结合位点附近。
我们首次报道了生化测量结果表明，功能测量实际上反映了与 NR2B 亚基 ATD 的结合。从这项研究中获得的见解有助于推进 ifenprodil 是 NR2B 亚基 ATD 的配体的理论。
