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The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


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.
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f2: 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.

Mentions: We introduced the four GluD2 binding site mutations one at a time into the GluD2-LBD for ITC experiments (Table 1, raw data and isotherms are presented in Supplementary Fig. S2). The Y496F and Y543Q mutations had only minor effects on D-serine binding affinity (Kd of 571 μM and 790 μM, Table 1) compared to wild-type GluD2-LBD (809 μM). The interaction of Tyr496 in GluD2 with D-serine does not involve the hydroxyl group of Tyr496, which may explain why the mutation to phenylalanine has little impact. The A686S mutation caused a slight reduction in binding affinity (Kd = 1090 μM, Table 1). All three D-serine-binding NMDA receptor subunits have a serine at the corresponding position that forms a hydrogen bond with D-serine1617. Introducing a serine residue into GluD2 at this position might therefore interfere in an unfavourable manner with the native hydrogen bonding network that involves the hydroxyl group of Tyr543 (Fig. 2a). The Y770F mutation had the largest impact, increasing D-serine affinity 7-fold compared to GluD2-LBD (Kd = 117 μM vs. 809 μM, Table 1). Remarkably, the mutation altered the sign of the enthalpy, making the binding exothermic (ΔH = −2 kcal/mol compared to 3 kcal/mol at wild-type GluD2-LBD), but at the same time reduced the favourable entropy (−TΔS = −3 kcal/mol compared to −7 kcal/mol at wild-type GluD2-LBD).


The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region
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.
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f2: 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.
Mentions: We introduced the four GluD2 binding site mutations one at a time into the GluD2-LBD for ITC experiments (Table 1, raw data and isotherms are presented in Supplementary Fig. S2). The Y496F and Y543Q mutations had only minor effects on D-serine binding affinity (Kd of 571 μM and 790 μM, Table 1) compared to wild-type GluD2-LBD (809 μM). The interaction of Tyr496 in GluD2 with D-serine does not involve the hydroxyl group of Tyr496, which may explain why the mutation to phenylalanine has little impact. The A686S mutation caused a slight reduction in binding affinity (Kd = 1090 μM, Table 1). All three D-serine-binding NMDA receptor subunits have a serine at the corresponding position that forms a hydrogen bond with D-serine1617. Introducing a serine residue into GluD2 at this position might therefore interfere in an unfavourable manner with the native hydrogen bonding network that involves the hydroxyl group of Tyr543 (Fig. 2a). The Y770F mutation had the largest impact, increasing D-serine affinity 7-fold compared to GluD2-LBD (Kd = 117 μM vs. 809 μM, Table 1). Remarkably, the mutation altered the sign of the enthalpy, making the binding exothermic (ΔH = −2 kcal/mol compared to 3 kcal/mol at wild-type GluD2-LBD), but at the same time reduced the favourable entropy (−TΔS = −3 kcal/mol compared to −7 kcal/mol at wild-type GluD2-LBD).

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.