Cryo-EM structures of the ionotropic glutamate receptor GluD1 reveal a non-swapped architecture

Abstract

Ionotropic orphan delta receptors (GluD) are not gated by glutamate or any other endogenous ligand but are grouped with ionotropic glutamate receptors based on sequence similarity. GluD1 receptors play critical roles in synaptogenesis, synapse maintenance and have been implicated in neuronal disorders including schizophrenia, cognitive deficits, and cerebral ataxia. Here we report cryo-electron microscopy structures of the rat GluD1 receptor complexed with calcium and the ligand 7-chlorokynurenic acid, elucidating molecular architecture and principles of receptor assembly. The structures reveal a non-swapped architecture at the extracellular amino-terminal (ATD) and ligand-binding domain (LBD) interface. This is in contrast to other families of ionotropic glutamate receptors (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.

The Architecture of GluD2 Ionotropic Delta Glutamate Receptor Elucidated by cryo-EM

“…However, the distances between the COMs of D1 and D2 domains of LBDs are ~25 Å and ~26 Å for subunits A-B and C-D respectively (Figure 4 C). This is similar to that observed in the GluD1 receptor structure determined in the presence of same ligands (1mM Ca 2+ and 7-CKA) (Burada et al, 2020)…”

“…Our cryo-EM analysis revealed a unique conformation of the tetrameric GluD2 receptor, whereby the Y-shaped arrangement of the extracellular ATD and LBD domains observed in AMPA, Kainate, and GluD1 receptors is disrupted (Figure 2 A). The two dimer arms made up of subunits AB and CD adopt an un-swapped architecture similar to that seen in GluD1 receptors (Burada et al, 2020). The dimer arm AB has two-fold symmetric arrangement at the ATD layer and a quasi-two-fold arrangement of the LBD layer similar to that observed in GluD1 receptors (Figure 2 B).…”

“…However, we cannot rule out the influence of grid preparation conditions and air-water interface interactions in observation of this unique asymmetric arrangement of the two-extracellular arms. Moreover in the absence of domain swapping, the conformational freedom for the movement of the two dimer arms is substantially higher as seen in the GluD1 receptors 15 and unlike that in AMPA 20 21 and kainate receptors 22 23 and we could have trapped a physiologically relevant conformational intermediate. It's also important to note that this conformation may not be compatible with trans-synaptic, tripartite complex formed between GluD2, cerebelin, and neurexin which is essential for maintaining the synaptic integrity of PF-PC synapses as this would limit the large-scale motions of the two extracellular arms.…”

“…The HEK293S GnTI -(ATCC CRL-3022) were obtained and authenticated by ATCC, and no further authentication or mycoplasma testing was performed. Expression and purification were carried out as reported earlier 15 Eluted fractions were analyzed for homogeneity by SDS-PAGE and FSEC, only fractions containing tetrameric receptors were pooled and concentrated to ~0.8 mg/ml.…”

“…The HEK293S GnTI -(ATCC CRL-3022) were obtained and authenticated by ATCC, and no further authentication or mycoplasma testing was performed. Expression and purification were carried out as reported earlier (Burada et al, 2020). Briefly, suspension adapted HEK293GnTIcells growing in freestyle 293 expression media supplemented with 2 % FBS (Gibco), 2 mM Glutamine (Gibco) and 1% Penstrep (Gibco) and were infected with high-titer baculovirus having the GluD2 construct at a multiplicity of infection (MOI) of ~1.…”