Project Summary The goal of this project is to unravel molecular details of pharmacologically active sites in N-methyl-D-aspartate receptors (NMDARs) with a scope to develop subtype-specific reagents against mental health-related disorders and diseases including depression, psychosis, schizophrenia, epilepsy, Alzheimer’s disease, and stroke where dysfunctional NMDARs are implicated. NMDARs belong to the ionotropic glutamate receptor (iGluR) family which mediates the majority of excitatory synaptic transmission in mammalian brains. They are hetero-multimeric ligand-gated ion channels composed of GluN1 and GluN2 and/or GluN3 subunits. The GluN1 and GluN3 subunits bind co-agonists including glycine and D-serine, whereas the GluN2 subunits bind glutamate and NMDA. Each NMDAR subunit contains an amino terminal domain (ATD), a ligand-binding domain (LBD), a transmembrane domain (TMD), and a carboxyl terminal domain (CTD). The GluN1-GluN2 NMDARs open their TMD ion channels upon binding of glycine and glutamate. NMDARs subtypes, defined by four distinct GluN2 subunits (A through D) or two distinct GluN3 (A and B) in combination with GluN1, exhibit different functional properties and spatio-temporal expression patterns. In the previous grant cycles, we unraveled the fragment structures of GluN1-2A and GluN1-2B ATDs, GluN1-2A and GluN1-2D LBDs, and intact structures of GluN1-2B by utilizing x-ray crystallography and single-particle electron cryo-microscopy (cryo-EM). Our findings provided molecular insights into channel assembly and basic patterns of conformational movements leading to activation, inhibition, and allosteric regulation. Despite these advances, there are still many fundamental questions remaining unanswered, including the completely unexplored structures of the GluN2C-containing NMDARs, unknown/uncharacterized allosteric and channel blockade sites with clinical relevance, and novel regulation by antibodies. We will now conduct research aimed at fulfilling these shortfalls. Aim 1 will determine the first structure of GluN2C-containing NMDARs; Aim 2 will identify and characterize pharmacological sites of clinically relevant compounds including ketamine, phencyclidine, memantine, and Glyx13. Aim 3 will unravel the mechanism of binding and functional regulation by novel engineered antibodies, and the auto-immune antibody for the first time. These three aims will be achieved by obtaining the structural information of intact NMDAR and domain fragments in the presence and absence of reagents by utilizing x-ray crystallography and single-particle cryo-EM. Structure-based mechanistic hypotheses will be examined mainly by electrophysiology. Successful completion of the proposed research aims will provide a detailed understanding about NMDAR subtypes- specificity, the regulatory mechanism by different reagents, and novel means to control NMDAR subtypes, which will help develop therapeutic strategies for neurological disorders and diseases.