Yi F , Mou TC , Dorsett KN , Volkmann RA , Menniti FS , Sprang SR , Hansen KB .

P20 GM103546 NIGMS NIH HHS , P41 GM103393 NIGMS NIH HHS , R01 NS097536 NINDS NIH HHS

	Figure 1. Glycine Sensitivity of Inhibition by GluN2A-Selective NAMs (A) Chemical structures of GluN2A-selective negative allosteric modulators (NAMs). (B) Representative recordings showing inhibition of diheteromeric GluN1/2A and triheteromeric GluN1/2A/2B receptors by NAMs. Responses are activated by 100 μM glutamate in the continuous presence of 3 μM glycine and recorded using two-electrode voltage-clamp electrophysiology. Triheteromeric GluN1/2AC1/2BAC2 receptors are expressed without diheteromeric GluN1/2A and GluN1/2B receptors using engineered C1 and C2 tags (Hansen et al., 2014). Horizontal and vertical scale bars represent 1 min and 100 nA, respectively. (C and D) NAM concentration-inhibition data at GluN1/2A receptors (C) comprised of two GluN1 and two GluN2A subunits and GluN1/2A/2B receptors (D) comprised of two GluN1 and two different GluN2 subunits (GluN2A and GluN2B). Inhibition is measured for responses activated by 100 μM glutamate in the continuous presence of 1, 3, 30, or 300 μM glycine. Data are mean ± SEM from 5 to 14 oocytes. See also Figures S1 and S2; Table S1.
	Figure 2. Structures of NAM-Bound GluN1/2A LBD Heterodimers (A) Structure of the NMDA receptor composed of two GluN1 and two GluN2 subunits (PDB: 4PE5; Karakas and Furukawa, 2014). One GluN1/GluN2 LBD heterodimer is highlighted in orange and yellow. (B) Crystal structure of the soluble glycine/glutamate-bound GluN1/2A LBD heterodimer in complex with TCN-201. Expression and purification of soluble NMDA receptor LBDs are made possible by deleting the amino-terminal domain (ATD) and replacing the transmembrane domain (TMD) with a Gly-Thr dipeptide linker. (C) NAM ligands from crystal structures of glycine/glutamate-bound GluN1/2A LBD heterodimers in complex with TCN-201 (2.28 à ), MPX-004 (2.52 à ), and MPX-007 (2.11 à ). The NAM ligands could be unambiguously fitted to their electron densities, shown as gray mesh (mFo-DFc omit map contoured at 2.5 σ). (D) Chemical structures of the NAMs, here exemplified by TCN-201, contain a halogenated ring A, a variable ring B, which is phenyl in TCN-201 and pyrazine in MPX-004/MPX-007, and a variable head group with a ring C that is phenyl in TCN-201 and thiazole in MPX-004/MPX-007. (E) Overlay of TCN-201 (gray), MPX-004 (cyan), and MPX-007 (salmon) from their respective GluN1/GluN2A LBD structures. (F–H) Views of the NAM binding site in structures of the glycine/glutamate-bound GluN1/2A LBD heterodimer in complex with TCN-201 (gray; F), MPX-004 (cyan; G), and MPX-007 (salmon; H). The NAM binding site is formed by residues from β sheet 10 and α helices J and K in GluN2A (yellow) and β sheets 10 and 14 in GluN1 (orange). See Supplemental Information for more detailed views of the NAM binding site. See also Figure S4 and Table S2.
	Figure 3. Analysis of NAM-Mediated Structural Changes in GluN1/2A LBD Structures (A) Structural alignment of the NAM binding site in the GluN1/2A LBD heterodimer without bound NAM (yellow and orange) and in complex with TCN-201 (gray), MPX-004 (cyan), and MPX-007 (salmon). (B) Difference distance maps showing pairs of Cα atoms that are less than 15 à apart in the NAM-lacking structure and change by more than 0.5 à in NAM-bound structures. The difference distance |ΔDij| is the absolute value of the distance measured in the NAM-lacking structure minus the distance measured in the NAM-bound structure. See also Figure S5.
	Figure 4. Relationship between NAM Inhibition and the GluN1 LBD Conformation (A) Cartoon illustrating conformations of the GluN1/2A LBD heterodimer with the NAM binding site in the subunit interface. In this cartoon, glutamate is continuously bound to the GluN2A LBD and glycine must induce a closed GluN1 LBD conformation for NMDA receptor activation. The cartoon also depicts the engineered disulfide bond introduced by N499C+Q686C mutations in GluN1 (GluN1-CC). (B) Representative two-electrode voltage-clamp recordings of responses from GluN1-CC/2A receptors with the GluN1 LBD locked in the closed, active conformation. Reponses are activated by 100 μM glutamate alone, followed by co-application of glycine (300 μM), TCN-201 (5 μM), MPX-004 (10 μM), or MPX-007 (30 μM). Horizontal and vertical scale bars represent 30 s and 50 nA, respectively. (C) Summary of the effects of glycine (300 μM), the competitive glycine site antagonist 7CKA (10 μM), TCN-201 (5 μM), MPX-004 (10 μM), and MPX-007 (30 μM) on glutamate-activated responses from GluN1-CC/2A receptors. Data are mean ± SEM from 5 to 6 oocytes. ∗ indicates significantly different from all other groups (p < 0.05; one-way ANOVA with Tukey-Kramer posttest). (D and E) Representative recordings of responses from GluN1-CC/2A receptors activated by 100 μM glutamate alone. DTT (4 mM) or 7CKA (10 μM) (D) did not affect responses, whereas co-application of DTT (4 mM) plus the competitive antagonist 7CKA (10 μM) or MPX-007 (30 μM) (E) diminished glutamate-activated responses. Horizontal and vertical scale bars represent 1 min and 100 nA, respectively. (F) Summary of the effects of DTT alone (4 mM) and co-application of DTT plus 7CKA (10 μM), TCN-201 (5 μM), MPX-004 (10 μM), or MPX-007 (30 μM) on glutamate-activated responses from GluN1-CC/2A receptors. Data are mean ± SEM from 5 to 24 oocytes See also Figure S6.
	Figure 5. Crystal Structures of Conformational States in the NAM Inhibition Cycle (A) Cartoon illustrating conformations of the GluN1/2A LBD heterodimer in the NAM inhibition cycle. In this cartoon, glutamate is continuously bound to the GluN2A LBD and the competitive glycine site antagonist DCKA stabilizes the open (i.e., glycine-lacking) state of the GluN1 LBD. PDB IDs of structures corresponding to the four conformations are listed. (B) Overlay of DCKA/glutamate-bound (PDB: 5JTY; yellow and orange) and glycine/glutamate-bound (PDB: 5I59; gray) structures in complex with MPX-007 illustrating the difference in GluN1 LBD domain closure (indicated by the arrow). (C) Crystal structures of the GluN1/2A LBD heterodimer corresponding to conformational states in the NAM inhibition cycle. Arrows indicate transitions between states and structural changes.
	Figure 6. Structural Changes in the NAM Binding Site during Allosteric Inhibition (A) Structural changes in modulatory binding site upon NAM binding to the glycine/glutamate-bound GluN1/2A LBD heterodimer. Binding of NAM (i.e., MPX-007) displaces GluN2A V783 and this “push” is accompanied by a steric effect on GluN1 F754. (B) Structural changes upon glycine unbinding from the NAM-bound GluN1/2A LBD heterodimer. Glycine unbinding and opening of the GluN1 LBD (here stabilized by DCKA) enable a conformational change of GluN1 F754, removing steric interaction with GluN2A V783. (C) Structural changes upon NAM unbinding from the DCKA/glutamate-bound GluN1/2A LBD heterodimer. NAM unbinding enables a relaxation of residues in the GluN1 hinge region. (D) Structural changes upon glycine binding to the NAM-lacking GluN1/2A LBD heterodimer. Glycine binding and closure of the GluN1 LBD reengages the steric interaction between GluN2A V783 and GluN1 F754 and places residues in the GluN1 hinge region in a conformation permissive for NAM binding. See also Figure S7.
	Figure 7. Mutational Analyses of the Influence of GluN2A V783 and GluN1 F754 on Glycine Potency (A) View of the NAM binding site in the glycine/glutamate-bound GluN1/2A LBD heterodimer in complex with MPX-007. The van der Waals radius of the methyl group of MPX-007 is shown as transparent sphere. This methyl group contacts the backbone carbonyl of GluN2A P527 and the side chain of GluN2A V783. There is also a nonpolar interaction between side chains of GluN2A V783 and GluN1 F754. (B) Glycine concentration-response data for wild-type and mutated GluN1/2A receptors. Responses are recorded in the presence of 100 μM glutamate using two-electrode voltage-clamp electrophysiology. Data are mean ± SEM from 9 to 16 oocytes. See also Table S3.
	Figure 8. Evaluation of GluN1 Y535 and F754 in Allosteric Inhibition of Glycine Binding (A) Operational model for TCN-201 inhibition by allosteric modulation of glycine binding without changing agonist efficacy (E). A is the agonist glycine, B is the NAM, and R is the receptor. Agonist binding affinity (KA) is changed by an allosteric constant α upon NAM binding, and NAM binding affinity (KB) is changed by α upon agonist binding. The equation for the dose ratio DR, the ratio of agonist EC50 values in presence and absence of modulator, is shown. (B) Glycine concentration-response data for GluN1/2A in the absence or presence of TCN-201 are analyzed by simultaneously fitting all data to both the dose ratio DR equation and the Hill equation using global nonlinear regression, yielding the glycine EC50, TCN-201 binding affinity (KB), and allosteric constant α that describe all the experimental data. (C) DR values derived from fitting individual concentration-response data shown in (B) are plotted as a function of TCN-201 concentration in the Schild plot, which also illustrates the effects of changing the allosteric constant α, but with constant KB (27 nM). The black line is generated using the KB and α obtained by fitting all data in (B) using global nonlinear regression (i.e., the black line is not obtained by fitting directly to DR values). Competitive antagonists have α = 0 (dashed line). (D–G) Schild analysis of TCN-201 inhibition for mutated GluN1/2A receptors with GluN1-Y535F (D), GluN1-Y535L (E), GluN1-F754L (F), or Glu-F754V (G) substitutions. Glycine concentration-response data are analyzed using global nonlinear regression (left side) and the resulting binding affinity (KB) and allosteric constant (α) are used to generate the black line in the Schild plot (right side) with the corresponding DR values. Dashed red line indicates the Schild plot for wild-type GluN1/2A receptors. See also Figures S1 and S8; Table S3.

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed
Akazawa, Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats. 1994, Pubmed
Benveniste, Kinetic analysis of antagonist action at N-methyl-D-aspartic acid receptors. Two binding sites each for glutamate and glycine. 1991, Pubmed
Bettini, Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors. 2010, Pubmed
Bissantz, A medicinal chemist's guide to molecular interactions. 2010, Pubmed
Borschel, Kinetic contributions to gating by interactions unique to N-methyl-D-aspartate (NMDA) receptors. 2015, Pubmed
Cheriyan, Pharmacology of triheteromeric N-Methyl-D-Aspartate Receptors. 2016, Pubmed
Edman, TCN 201 selectively blocks GluN2A-containing NMDARs in a GluN1 co-agonist dependent but non-competitive manner. 2012, Pubmed , Xenbase
Emsley, Features and development of Coot. 2010, Pubmed
Furukawa, Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core. 2003, Pubmed
Furukawa, Subunit arrangement and function in NMDA receptors. 2005, Pubmed
Gielen, Mechanism of differential control of NMDA receptor activity by NR2 subunits. 2009, Pubmed
Hackos, Positive Allosteric Modulators of GluN2A-Containing NMDARs with Distinct Modes of Action and Impacts on Circuit Function. 2016, Pubmed
Hansen, Distinct functional and pharmacological properties of Triheteromeric GluN1/GluN2A/GluN2B NMDA receptors. 2014, Pubmed , Xenbase
Hansen, Structural determinants of agonist efficacy at the glutamate binding site of N-methyl-D-aspartate receptors. 2013, Pubmed , Xenbase
Hansen, Subunit-selective allosteric inhibition of glycine binding to NMDA receptors. 2012, Pubmed
Hatton, Modulation of triheteromeric NMDA receptors by N-terminal domain ligands. 2005, Pubmed , Xenbase
Ishii, Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits. 1993, Pubmed , Xenbase
Jespersen, Structural insights into competitive antagonism in NMDA receptors. 2014, Pubmed , Xenbase
Karakas, Crystal structure of a heterotetrameric NMDA receptor ion channel. 2014, Pubmed
Karakas, Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors. 2011, Pubmed , Xenbase
Kazi, Mechanical coupling maintains the fidelity of NMDA receptor-mediated currents. 2014, Pubmed
Kussius, NMDA receptors with locked glutamate-binding clefts open with high efficacy. 2010, Pubmed
Lee, NMDA receptor structures reveal subunit arrangement and pore architecture. 2014, Pubmed , Xenbase
McCoy, Phaser crystallographic software. 2007, Pubmed
Monyer, Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. 1994, Pubmed
Otwinowski, Processing of X-ray diffraction data collected in oscillation mode. 1997, Pubmed
Paoletti, NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. 2013, Pubmed
Pierson, GRIN2A mutation and early-onset epileptic encephalopathy: personalized therapy with memantine. 2014, Pubmed , Xenbase
Rauner, Triheteromeric NR1/NR2A/NR2B receptors constitute the major N-methyl-D-aspartate receptor population in adult hippocampal synapses. 2011, Pubmed
Sheng, Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. 1994, Pubmed
Stroebel, Controlling NMDA receptor subunit composition using ectopic retention signals. 2014, Pubmed , Xenbase
Tovar, Triheteromeric NMDA receptors at hippocampal synapses. 2013, Pubmed
Traynelis, Glutamate receptor ion channels: structure, regulation, and function. 2010, Pubmed
Vicini, Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors. 1998, Pubmed
Volgraf, Discovery of GluN2A-Selective NMDA Receptor Positive Allosteric Modulators (PAMs): Tuning Deactivation Kinetics via Structure-Based Design. 2016, Pubmed
Volkmann, MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit. 2016, Pubmed , Xenbase
Williams, Ifenprodil discriminates subtypes of the N-methyl-D-aspartate receptor: selectivity and mechanisms at recombinant heteromeric receptors. 1993, Pubmed , Xenbase
Yao, Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals subtype-specific characteristics. 2013, Pubmed
Yuan, Functional analysis of a de novo GRIN2A missense mutation associated with early-onset epileptic encephalopathy. 2014, Pubmed
Yuan, Control of NMDA receptor function by the NR2 subunit amino-terminal domain. 2009, Pubmed , Xenbase
Yuan, Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases. 2015, Pubmed