Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, Shanghai, China.
Ecole Normale Supérieure, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Paris, France.
Sci Rep. 2016 Oct 7;6:34751. doi: 10.1038/srep34751.
Allostery is essential to neuronal receptor function, but its transient nature poses a challenge for characterization. The N-terminal domains (NTDs) distinct from ligand binding domains are a major locus for allosteric regulation of NMDA receptors (NMDARs), where different modulatory binding sites have been observed. The inhibitor ifenprodil, and related phenylethanoamine compounds specifically targeting GluN1/GluN2B NMDARs have neuroprotective activity. However, whether they use differential structural pathways than the endogenous inhibitor Zn for regulation is unknown. We applied genetically encoded unnatural amino acids (Uaas) and monitored the functional changes in living cells with photo-cross-linkers specifically incorporated at the ifenprodil binding interface between GluN1 and GluN2B subunits. We report constraining the NTD domain movement, by a light induced crosslinking bond that introduces minimal perturbation to the ligand binding, specifically impedes the transduction of ifenprodil but not Zn inhibition. Subtle distance changes reveal interfacial flexibility and NTD rearrangements in the presence of modulators. Our results present a much richer dynamic picture of allostery than conventional approaches targeting the same interface, and highlight key residues that determine functional and subtype specificity of NMDARs. The light-sensitive mutant neuronal receptors provide complementary tools to the photo-switchable ligands for opto-neuropharmacology.
变构作用对于神经元受体功能至关重要，但它的瞬态性质给其特征描述带来了挑战。与配体结合结构域不同的 N 端结构域（NTD）是 NMDA 受体（NMDAR）变构调节的主要部位，在那里已经观察到不同的调节性结合位点。抑制剂ifenprodil 和相关的苯乙胺化合物特异性靶向 GluN1/GluN2B NMDAR 具有神经保护活性。然而，它们是否使用不同于内源性抑制剂 Zn 的差异结构途径进行调节尚不清楚。我们应用了遗传编码的非天然氨基酸（Uaas），并通过专门在 GluN1 和 GluN2B 亚基之间的 ifenprodil 结合界面处掺入的光交联剂在活细胞中监测功能变化。我们报告说，通过光诱导的交联键限制了 NTD 结构域的运动，该交联键对配体结合的干扰最小，特异性地阻碍了 ifenprodil 的转导，但不阻碍 Zn 的抑制。在调节剂存在下，微小的距离变化揭示了界面的灵活性和 NTD 的重排。与针对相同界面的传统方法相比，我们的结果呈现出更丰富的变构作用动态图，并突出了决定 NMDAR 功能和亚型特异性的关键残基。光敏感突变神经元受体为光控神经药理学提供了与光开关配体互补的工具。
