Conformational rearrangement of the NMDA receptor amino-terminal domain during activation and allosteric modulation

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability. N-Methyl-D-aspartate receptors (NMDARs) activation involves closure of the GluN1 and GluN2 subunit ligand binding domains, which is regulated allosterically by the amino-terminal domain (ATD). Here, smFRET, used to monitor conformational rearrangements of the NMDAR ATD, reveals that glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation that are regulated by protons.