Subtype-specific conformational landscape of NMDA receptor gating

Abstract

N-methyl-D-aspartate receptors are ionotropic glutamate receptors that mediate synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties. To understand this diversity, we use single-molecule fluorescence resonance energy transfer (smFRET) to measure the conformations of the ligand binding domain and modulatory amino-terminal domain of the common GluN1 subunit in receptors with different GluN2 subunits. Our results demonstrate a strong influence of the GluN2 subunits on GluN1 rearrangements, both in non-agonized and partially agonized activation intermediates, which have been elusive to structural analysis, and in the fully liganded state. Chimeric analysis reveals structural determinants that contribute to these subtype differences. Our study provides a framework for understanding the conformational landscape that supports highly divergent levels of activity, desensitization, and agonist potency in receptors with different GluN2s and could open avenues for the development of subtype-specific modulators.

Keywords: CP: Neuroscience; FRET; GluN1; GluN2; Grin1; Grin2; NMDA receptors; allostery; single-molecule FRET.

Figure 1.. Agonists drive convergence of GluN1 conformation from GluN2-dependent resting FRET states

(A) Analysis of existing structural data (red = 0 structures, green = 1 or more) for NMDA receptors GluN1/2A-D in apo-like conditions (GluN1 site/GluN2 site: apo/apo, 5,7-dichlorokynurenic acid [DCKA]/D-2-amino-5-phosphonovalerate [D-APV], L689,560/SDZ 220–040), partially liganded with glycine (GluN1 site/GluN2 site: glycine/apo, glycine/SDZ 220–040, glycine/CPP), partially liganded with glutamate (GluN1 site/GluN2 site: apo/glutamate, L689,560/glutamate, CGP78608/glutamate), and fully liganded (GluN1 site/GluN2 site: glycine/glutamate). (B) Spheres in structures (PDB: 7EOS ) and stars in cartoons indicating labeling sites in D2 lobe of GluN1 (gray) LBD (D677) and R1 lobe of GluN1 NTD in NMDA receptors also containing GluN2 (transparent black in structure, black in cartoon). (C–F) Ligand-dependent conformations determined from intersubunit FRET between GluN1 LBDs with fluorophores incorporated at site D677TAG (C and E) or GluN1 NTDs with fluorophores incorporated at site W56TAG (D and F) in an apo-like condition (zero added glycine, 3 μM GluN1 antagonist CGP78608, zero added glutamate) (A and B) or with saturating concentrations of agonists (100 μM glycine, 1 mM glutamate) (C and D) when GluN1 is combined with GluN2A (orange), GluN2B (blue), GluN2C (teal), or GluN2D (magenta). (Left) Histograms of smFRET distributions indicating mean and SEM (error bars) across technical replicates of n = 4 movies or combined movies with total particle number across all movies listed in Table S1. (Right) Example smFRET traces with colors corresponding to histogram keys. Labeling sites indicated with green and red stars on cartoons. Donor (AF555) and acceptor (AF647) dyes were imaged at 10 fps.

Figure 2.. GluN2-dependent regulation of GluN1 conformation in single agonists

Ligand-dependent conformations determined from intersubunit FRET between GluN1(D677TAG) lower LBD (A–D) or GluN1(W56TAG) NTD (E–H) paired with GluN2A (A and E), GluN2B (B and F), GluN2C (C and G), or GluN2D (D and H) in 3 μM CGP78608 and 1 mM glutamate (purple) or 100 μM glycine and zero added glutamate (blue) or in apo-like state (zero added glycine, 3 μM GluN1 antagonist CGP, zero added glutamate) (green) or saturating agonists (100 μM glycine, 1 mM glutamate) (navy) reproduced from Figure 1. (Top) Histograms of smFRET distributions indicating mean and SEM (error bars for those histograms not shown in Figure 1) across technical replicates of n = 4 movies or combined movies with total particle number across all movies listed in Table S1. Labeling sites indicated with green and red stars on cartoon insets (A and E). (Bottom) Quantification of the spread of the distributions in above histograms using the same color scheme. For each, the left and right vertical ticks indicate the first and third quartiles and the middle the median. Donor (AF555) and acceptor (AF647) dyes were imaged at 10 fps. See also Figures S3 and S4.

Figure 3.. GluN2B/2D chimeras reveal structural determinants of subtype-specific NTD conformational dynamics

(A) GluN1a/GluN2B glycine/glutamate structure (PDB: 7SAA ) showing the regions swapped in chimeric GluN2 receptors. (B and C) Histograms of smFRET distributions indicating mean and SEM (error bars) across technical replicates of n = 4 individual movies or combined movies for ligand-dependent conformations determined from intersubunit FRET between GluN1(W56TAG) NTD paired with chimeric GluN2 subunits (according to scheme in Figure S5), which transplant pieces of GluN2D into GluN2B, focusing on the NTD (B) and LBD (C) in 3 μM CGP78608 and 1 mM glutamate (purple) and apo-like (zero added glycine, in 3 μM CGP78608; green) conditions. Labeling sites are indicated with green and red stars on cartoon insets at top right of (B) and (C). Total receptor number for each condition is listed in Table S1. Donor (AF555) and acceptor (AF647) dyes were imaged at 10 fps. See also Figure S5.

Figure 4.. GluN1 loop 2 deletion affects glutamate-bound NTD conformation in GluN2B, but not GluN2D, receptors

(A) GluN2 α5 helix (magenta) and residues 489–496 of GluN1 loop 2 (red) in the GluN1a/GluN2B glycine/glutamate structure (PDB: 7SAA ). (B and C) Histograms of smFRET distributions indicating the mean and SEM (error bars) across technical replicates of n = 4 individual movies or combined movies for ligand-dependent conformations determined from intersubunit FRET between GluN1(W56TAG,R489-K496GG) (solid) or GluN1(W56TAG) (dotted) NTD paired with GluN2B (B) or GluN2D (C) in 3 μM CGP78608 and 1 mM glutamate (purple) and apo-like (zero added glycine, in 3 μM CGP78608) conditions. Labeling sites are indicated with green and red stars on cartoons. Total receptor number for each condition is listed in Table S1. Donor (AF555) and acceptor (AF647) dyes were imaged at 10 fps.

Figure 5.. Subtype-specific conformational landscape of NMDA receptor gating

(A) Cartoon representations of GluN1 subunits within NMDA receptor heterotetramers containing different GluN2 subunits (not depicted) based on smFRET distributions in ligand conditions representing steps in the activation pathway. Where multiple versions of a single domain are represented, opacity indicates relative occupancy. Black dots indicate locations of FRET probes and lines the distance between probes. Structural regions that were determined to be important for particular subtype-specific conformations are indicated, though they may not be the unique determinants. (B) Comparison of conformations deduced based on inter-GluN1 FRET and structurally observed conformations. (Top row) Cartoons of GluN1 in different conformations based on smFRET data. (Bottom three rows) Structures of GluN2A(black)/GluN1(gray) receptors in splayed-open (PDB: 6MMI; glycine/glutamate/1 μM ZnCl2 [pH 7.4]), 1-Knuckle (PDB: 6MM9; glycine/glutamate/1 mM ZnCl2 [pH 6.1]), and 2-Knuckle (PDB: 6MMP; glycine/glutamate/0.1 mM EDTA [pH 8.0]) conformations with W56 and D677 shown as spheres. Labeled lines indicate distances between β-carbons of GluN1 residues W56 in the NTD and D677 in the LBD. (Bottom row) Top-down view of the NTD showing (left to right) zero, one, and two alpha helices forming the tetrameric interface between GluN2 subunits.