Ionotropic orphan delta (GluD) receptors are not gated by glutamate or any other endogenous ligand but are grouped with ionotropic glutamate receptors (iGluRs) based on sequence similarity. GluD1 receptors play critical roles in synaptogenesis and synapse maintenance and have been implicated in neuronal disorders, including schizophrenia, cognitive deficits, and cerebral ataxia. Here we report cryo-EM structures of the rat GluD1 receptor complexed with calcium and the ligand 7-chlorokynurenic acid (7-CKA), elucidating molecular architecture and principles of receptor assembly. The structures reveal a non-swapped architecture at the interface of the extracellular amino-terminal domain (ATD) and the ligand-binding domain (LBD). This finding is in contrast with structures of other families of iGluRs, where the dimer partners between the ATD and LBD layers are swapped. Our results demonstrate that principles of architecture and symmetry are not conserved between delta receptors and other iGluRs and provide a molecular blueprint for understanding the functions of the ‘orphan’ class of iGluRs.
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The cryo-EM density reconstruction and final models were deposited in the Electron Microscopy DataBase (accession code EMD-0744 for the compact conformation and EMD-0773 for the splayed conformation) and in the Protein Data Bank (accession code PDB 6KSS for the compact conformation and PRB 6KSP for the splayed conformation). The raw movie data have been submitted to the EMPIAR database. All other relevant data supporting the key findings of this study are available within the article and its Supplementary Information files or from the corresponding author upon reasonable request. Source data for Fig. 3a–c and Extended Data Fig. 9e are available with the paper online.
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This work was supported by the Wellcome Trust DBT India Alliance fellowship (grant number IA/I/13/2/501023) awarded to J.K. A.P.B. thanks ICMR (3/1/3/JRF-2014/HRD-86(60237), India for a senior research fellowship. R.V. thanks SERB for an N-PDF fellowship (N-PDF/2016/002621). M. L. Mayer, NIH, Bethesda kindly gifted the various iGluR constructs that were subcloned and used for construct optimization and mutational studies. E. Gouaux (OHSU, Portland) kindly provided the pEG BacMam vector. We acknowledge the European Synchrotron Radiation Facility for provision of microscope time on CM01, and we thank M. Hons for assistance in EM data collection. We thankfully acknowledge the kind help of M. Karuppasamy, EMBL in grid vitrification. Help with initial screening of conditions for grid vitrification from P. J. Peters, R. Ravelli at M4i, Maastricht University, Masstricht, the Netherlands and V. K. Ragunath, National Electron Cryo-Microscopy facility at the Bangalore Life Sciences Cluster (DBT/PR12422/MED/31/287/2014), NCBS, Bangalore is gratefully acknowledged. We are also thankful to A. Kembhavi and K. Vaghmare, The Inter-University Centre for Astronomy and Astrophysics, Pune for helping with transfer and storage of raw EM data.
The authors declare no competing interests.
Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Schematic representation of the optimized GluD1 construct showing the C-terminal truncation at residue 851 and C-terminal thrombin cleavage site, along with GFP and octa-histidine tag (Supplementary Notes). b, Size-exclusion profile of the final purified protein showing receptor stability in optimized buffer conditions. c, Selected 2D class averages from reference-free 2D classification of GluD1 in complex with 1 mM 7-CKA and 1 mM Ca2+. The white arrows mark a few classes that show conformational heterogeneity of the extracellular receptor domains. d, 3D classification of GluD1 into seven classes reveals heterogeneity due to the movement of the two extracellular arms. More details are in Extended Data Fig. 2 and Supplementary Fig. 1.
A total of 72,149 good particles were obtained by several cycles of 2D class averaging of particles from 4,120 micrographs. The 3D map generated by ab initio 3D reconstruction was further refined heterogeneously into seven conformationally distinct 3D classes. The 3D classes showed heterogeneity due to movement of the two extracellular arms. For the purpose of model building and analysis, a compact (class 5) and a splayed (class 7) conformation map were further refined to a resolution of 8.1 Å and 7.6 Å, respectively.
a, d, The sharpened cryo-EM densities of GluD1 Δ851 in 7-CKA and calcium bound form, colored based on local resolution. b, e, Euler angle distribution of particles for the two models is shown. c, f, FSC curves for the cryo-EM maps with mask (red) without mask (blue). The resolution of map corresponding to FSC 0.5 and 0.143 is indicated.
a−f, Architecture of splayed conformation of GluD1 receptors in complex with 7-CKA and calcium. a, Side view highlighting the broadest face of the Y-shaped receptor and 90° rotated views of the sharpened 3D density map. Each subunit is depicted in a different color. The EM reconstructions clearly show the non-swapped arrangement of the ATD and LBD layers. The distances between the centroids (R1-R1 of ATD domains) for AB and CD dimer pairs are shown above the model. The vertical separation between the COMs of ATD dimers and LBD dimers are also shown. Panel b shows the segmented density map for subunits A and B fitted with protein coordinates. c, Superimposition of subunits B/D, B/C, A/D and A/C are shown highlighting similar AB and BC conformations. Helices and sheets are represented as pipes and planks, respectively. Top views of ATD (d), LBD (e) and TM domains (f) are shown. The distances and the angles subtended between the COM of various subunits were measured and are indicated below the top views. More details are in Extended Data Figs. 5 and 6.
a, Cryo-EM density map of compact GluD1 model is shown in a view parallel to the membrane. The four subunits A, B, C, D are colored in orange, green, yellow and cyan, respectively. The colored spheres represent the COM of the ATD and LBD domains. b, Top view of ATD with segmented EM density map fitted with atomic models is shown. The distances between the COMs of ATDs shown with dashed lines below the EM density map, depicting the arrangement of ATDs in the plane. c, Densities corresponding to LBDs fitted with atomic models. d, The distances from COMs of ATD and LBD are shown. The LBD plane is depicted as a circular disk, and the ATD plane is shown as a dashed ellipse. e,f, Side and top views of angles subtended by COMs of ATD with the COM of the LBD layer. COM plane of the LBD layer is indicated by metallic disk.
a, Cryo-EM density map of splayed GluD1 model is shown in a view parallel to the membrane. The four subunits A, B, C, D are colored in orange, green, yellow and cyan, respectively. The colored spheres represent the COM of ATD and LBD domains. b, Top view of ATD with segmented EM density map fitted with atomic models is shown. The distances between the COMs of ATDs are shown with dashed lines below the EM density map, depicting the arrangement of ATDs in the plane. c, Densities corresponding to LBDs fitted with atomic models is shown. d, The distances from COMs of ATD and LBD are shown. The LBD plane is depicted as a circular disk, and the ATD plane is shown as a dashed ellipse. e,f, Side and top views of angles subtended by COMs of ATD with the COM of the LBD layer. COM plane of the LBD layer is indicated by metallic disk.
Extended Data Fig. 7 Domain arrangement in GluD1, GluA2, GluK2, GluN1/GluN2A and GluN1/GluN2B receptors.
a, Top views of the ATD (a), LBD (b) and TM domains (c) are shown for GluD1, GluA2, GluK2, GluN1/GluN2A and GluN1/GluN2B receptors highlighting the subunit arrangement. Each chain is uniquely colored, and domain arrangement is also depicted in cartoon below each layer. Comparisons for compact and super-splayed conformations of NMDA receptors with that of GluD1 are shown highlighting the fact that in all the conformations of AMPA, kainate and NMDA receptors, the domain swapping between the ATD and LBD layers exists unlike that in GluD1.
Surface illustration of the isolated subdomains in gray with buried surface represented in green. The calculated buried surface area for the various domains is also shown. a–d, Analysis for ATD dimer, LBD dimer, ATD dimer-of-dimer and LBD dimer-of-dimer interface for the compact GluD1 model.
a, Representative traces for the whole-cell recording of the GluD1, GluD1 Δ851, GluD1(K2LBD) and GluD1(K2LBD) Δ851 expressed in HEK-293T cells are shown in response to 10 mM glutamate application. b–e, Whole-cell patch-clamp recordings (holding potential = −60 mV) from constitutively active GluD1 A634C point mutant receptors. The seal resistance before entering into the whole-cell configuration was always at least 1 GΩ. b, No spontaneous currents were observed for wild-type GluD1 or GluD1 Δ851 receptors, and no effect was observed on 2 mM Ca2+ application. c,d, Overlay of representative traces showing application of either NMDG solution or 1 mM 7-CKA (red), 10 mM d-serine (green) or 2 mM CaCl2 (blue). Dashed line indicates zero current level achieved by application of impermeant NMDG, which blocks the constitutive inward currents for both GluD1 A634C (c) and GluD1 Δ851 A634C receptors (d). The constitutive currents are also modestly inhibited by D-ser or 7-CKA application and potentiated by Ca2+ (c,d) for both the full-length and CT truncated GluD1 receptors. e, Percent inhibition of spontaneous currents by 7-CKA and d-serine calculated with respect to NMDG inhibition. Data for graphs are available as Source data. The number of cells used for the recordings is shown. The error bars represent standard error from the mean.
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Burada, A.P., Vinnakota, R. & Kumar, J. Cryo-EM structures of the ionotropic glutamate receptor GluD1 reveal a non-swapped architecture. Nat Struct Mol Biol 27, 84–91 (2020). https://doi.org/10.1038/s41594-019-0359-y