SUMMARY The physiology of N-Methyl-D-aspartate (NMDA) receptors in mammals is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino terminal domain (ATD). Recent crystal structures of GluN1/GluN2B NMDA receptors in the presence of agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here, we combine x-ray crystallography, single-particle electron cryomicroscopy, and electrophysiology to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open-conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel.

Summary There has been a great level of enthusiasm to down-regulate overactive N-methyl-d-aspartate (NMDA) receptors to protect neurons from excitotoxicity. NMDA receptors play pivotal roles in basic brain development and functions as well as in neurological disorders and diseases. However, mechanistic understanding of antagonism in NMDA receptors is limited due to complete lack of antagonist-bound structures for the l-glutamate-binding GluN2 subunits. Here we report the crystal structures of GluN1/GluN2A NMDA receptor ligand-binding domain (LBD) heterodimers in complex with GluN1- and GluN2-targeting antagonists. The crystal structures reveal that the antagonists, D-(−)-2-Amino-5-phosphonopentanoic acid (d-AP5) and 1-(Phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA), have discrete binding modes and mechanisms for opening of the bilobed architecture of GluN2A LBD compared to the agonist-bound form. The current study shows distinct ways by which the conformations of NMDA receptor LBDs may be controlled and coupled to receptor inhibition and provides possible strategies to develop therapeutic compounds with higher subtype-specificity.

N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels assembled from GluN1 and GluN2 subunits. We used a series of N-hydroxypyrazole-5-glycine (NHP5G) partial agonists at the GluN2 glutamate binding site as tools to study activation of GluN1/GluN2A and GluN1/GluN2D NMDA receptor subtypes. Using two-electrode voltage-clamp electrophysiology, fast-application patch-clamp, and single-channel recordings, we show that propyl-and ethyl-substituted NHP5G agonists have a broad range of agonist efficacies relative to the full agonist glutamate (,1-72%). Crystal structures of the agonist binding domains (ABDs) of GluN2A and GluN2D do not reveal any differences in the overall domain conformation induced by binding of the full agonist glutamate or the partial agonist propyl-NHP5G, which is strikingly different from ABD structures of 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propanoate (AMPA) and kainate receptors bound to full and partial agonists. Subsequent evaluation of relative NHP5G agonist efficacy at GluN2A-GluN2D chimeric subunits implicates the amino-terminal domain (ATD) as a strong determinant of agonist efficacy, suggesting that interdomain interactions between the ABD and the ATD may be a central element in controlling the manner by which agonist binding leads to channel opening. We propose that variation in the overall receptor conformation, which is strongly influenced by the nature of interdomain interactions in resting and active states, mediates differences in agonist efficacy and partial agonism at the GluN2 subunits.

Highlights d Structures of GluN1b-GluN2B NMDARs in distinct states were solved at 4 Å or better d Agonist-binding ''stretches'' the GluN2B LBD-TMD linkers to perturb the channel gate d A GluN2 antagonist mediates inhibition by relaxing the GluN2 LBD-TMD linkers d A GluN1 antagonist reorients GluN1-GluN2 dimers to relax the GluN2 LBD-TMD linkers

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