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The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region.
Tapken D
,
Steffensen TB
,
Leth R
,
Kristensen LB
,
Gerbola A
,
Gajhede M
,
Jørgensen FS
,
Olsen L
,
Kastrup JS
.
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Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine.
Figure 1. Structure of the GluD2-LBD (PDB ID 2V3U13).(a) The D1–D2 hinge region (HS1 and HS2; pink) as well as the positions of point mutations in the binding site generated in this study (Y496F: purple; Y543Q: pink; A686S: blue; Y770F: brown) are indicated. The box on the right shows a magnified view of the D1–D2 hinge region and the D-serine binding site with all residues investigated in this study labelled. (b) Comparison of HS1 and HS2 residues among all 18 iGluR subunits. Residues conserved among all subunits are highlighted in magenta, residues conserved within subfamilies in cyan.
Figure 2. Potencies and efficacies of D-serine at GluD2-Lc binding site mutants.(a) Binding site of GluD2 (PDB ID 2V3U13) with the four residues mutated in this study shown in stick representation (Y496: purple; Y543: light red; A686: blue; Y770: brown). (b) Concentration–response curves for inhibition by D-serine of spontaneous currents mediated by GluD2-Lc and its binding site mutants determined by two-electrode voltage clamp electrophysiology. Each data point is shown as mean ± SEM from 8–11 oocytes. (c) Potencies of D-serine at GluD2-Lc and its binding site mutants displayed as IC50 values calculated from the concentration–response curves shown in b. Data are shown as means ± SEM from 8–11 oocytes. The significances of differences from GluD2-Lc were calculated for the logIC50 values by one-way ANOVA followed by Dunnett’s multiple comparisons test; ns, P > 0.05; ****P ≤ 0.0001. (d) Efficacies of D-serine at GluD2-Lc and its binding site mutants, calculated as the fraction of the spontaneous current that is inhibited by D-serine. Data are shown as means ± SEM from 20–30 oocytes. The significances of differences from GluD2-Lc were calculated by one-way ANOVA followed by Dunnett’s multiple comparisons test; ns, P > 0.05; ****P ≤ 0.0001. (e) Representative spontaneous currents of GluD2-Lc and its binding site mutants determined by switching from Na+-free to Na+-containing extracellular solution and their inhibition by D-serine application.
Figure 3. Potencies and efficacies of D-serine at GluD2-Lc D1–D2 hinge region mutants.(a) Concentration–response curves for inhibition by D-serine of spontaneous currents mediated by GluD2-Lc, its full hinge region mutant GluD2-Lc(H)GluN1, and three partial hinge region mutants that respond to D-serine. The concentration–response curve for activation of GluN1-1a/GluN2A by D-serine in the presence of 100 μM glutamate is shown for comparison. All curves were determined by two-electrode voltage clamp electrophysiology. Each data point is shown as mean ± SEM from 6–11 oocytes. (b) Potencies of D-serine at GluD2-Lc and its full and partial hinge region mutants displayed as IC50 values calculated from the concentration–response curves shown in a. Data are shown as means ± SEM from 6–11 oocytes. The significances of differences from GluD2-Lc were calculated for the logIC50 values by one-way ANOVA followed by Dunnett’s multiple comparisons test; ns, P > 0.05; ****P ≤ 0.0001. (c) Current traces of GluD2-Lc and GluD2-Lc-(H)GluN1 upon application of various D-serine concentrations. The traces are normalized to the amplitude of the current induced by a saturating D-serine concentration (10 mM for GluD2-Lc, 10 μM for GluD2-Lc-(H)GluN1). (d) Current traces of GluD2-Lc and GluD2-Lc-(H)GluN1 recorded at high D-serine concentrations. GluD2-Lc-(H)GluN1 shows peak and tail currents upon application and removal of D-serine, respectively. (e) Efficacies of D-serine at GluD2-Lc and its full and partial hinge region mutants, calculated as the fraction of the spontaneous current that is inhibited by D-serine. Data are shown as means ± SEM from 20–30 oocytes. The significances of differences from GluD2-Lc were calculated by one-way ANOVA followed by Dunnett’s multiple comparisons test; **P ≤ 0.01; ****P ≤ 0.0001. (f) Representative spontaneous currents of GluD2-Lc and its hinge region mutants determined by switching from Na+-free to Na+-containing extracellular solution and their inhibition by D-serine application.
Figure 4. Molecular dynamics simulations on GluD2-LBD, GluN1-LBD, and GluD2-LBD-(H)GluN1 in their D-serine-bound forms.(a) Hydrogen bonds (yellow dashed lines) between D-serine (green) and binding site residues (sand) as well as from Asp742 to Tyr543 and Tyr770 (orange dashed lines) in GluD2-LBD. All hydrogen bonds to D-serine observed during the simulation have been mapped onto the X-ray structure (PDB ID 2V3U). (b) Hydrogen bonds between D-serine and binding site residues as well as from Gln536 to Asp732 and Ser756 (orange dashed lines) in GluN1-LBD. All hydrogen bonds to D-serine observed during the simulation have been mapped onto the X-ray structure (PDB ID 1PB8). (c) Hydrogen bonds between D-serine and binding site residues as well as from Gln543 to Asp742, Ser768, and Tyr770 in GluD2-LBD-(H)GluN1. All hydrogen bonds to D-serine observed during the simulation have been mapped onto the model structure. (d–f) Variation of the phi and psi torsional angles for two residues in the HS2 region during the 100 ns MD simulation. (d) GluD2-LBD with D-serine. (e) GluN1-LBD with D-serine. (f) GluD2-LBD-(H)GluN1 with D-serine. For phi and psi values of all the residues in HS2, see Supplementary Fig. S7.
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