Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 59812.
Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812.
Proc Natl Acad Sci U S A. 2017 Aug 15;114(33):E6942-E6951. doi: 10.1073/pnas.1707752114. Epub 2017 Jul 31.
NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A-D). We describe highly potent ()-5-[()-2-amino-2-carboxyethyl]-4,5-dihydro-1-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.
NMDA 型谷氨酸受体是配体门控离子通道，在中枢神经系统（CNS）中参与兴奋性神经传递。大多数 NMDA 受体由两个甘氨酸结合的 GluN1 和两个谷氨酸结合的 GluN2 亚基（GluN2A-D）组成。我们描述了高活性的（）-5-[()-2-氨基-2-羧乙基]-4,5-二氢-1-吡唑-3-羧酸（ACEPC）竞争性 GluN2 拮抗剂，其中 ST3 在 GluN1/2A 上的结合亲和力为 52 nM，在 GluN1/2B 受体上的结合亲和力为 782 nM。与广泛使用的 GluN2A 选择性拮抗剂 NVP-AAM077 相比，ST3 对 GluN1/2A 的 15 倍选择性得到了改善，我们发现 NVP-AAM077 对 GluN1/2A 的选择性是 GluN1/2B 的 11 倍。与结合 ACEPC 拮抗剂的 GluN1/2A 激动剂结合域（ABD）异源二聚体的晶体结构揭示了一种结合模式，其中配体占据延伸到 GluN1 和 GluN2A ABD 之间亚基界面的腔。突变分析表明，ST3 对 GluN2A 的偏好主要由四个不保守的残基介导，这些残基不直接与配体接触，但位于谷氨酸结合位点 12 Å 内。其中两个残基以有利于与 GluN2A 结合但阻止与 GluN2B 结合的方式影响 ST3 占据的腔。因此，我们通过鉴定 GluN2A 和 GluN2B 亚基之间存在差异的配体结合腔，揭示了设计亚基选择性竞争性 NMDA 受体拮抗剂的机会。这种结构上的见解表明，除了接触残基对结合亲和力的直接贡献外，谷氨酸结合位点拮抗剂的亚基选择性还可以通过其他机制介导。
