ELUCIDATING THE ARCHITECTURE OF ORPHAN GLUTAMATE DELTA RECEPTOR-1 BY CRYO-EM

Orphan glutamate delta (GluD) receptors play vital roles in normal brain functions and are associated with multiple neuronal disorders. Single-particle cryo-electron microscopy (cryo-EM) was used to determine the first structure of a full-length GluD1 receptor.

STRUCTURAL BIOLOGY

56 ESRF

Glutamate delta receptor ion channels (GluD) are the orphan cousins of AMPA, NMDA, and Kainate receptors and belong to the ionotropic glutamate receptor (iGluR) family. However, unlike its kin, GluD receptors are not gated by glutamate or any other known endogenous ligands, thus attracting the name orphan . Nonetheless, they play essential roles in synaptogenesis and synaptic plasticity, and are linked with several nervous system disorders such as schizophrenia, cognitive deficits, cerebral ataxia, and psychiatric disorders [1]. The GluD receptors are believed to mediate their functions by both canonical ionotropic and non- canonical metabotropic mechanisms. Moreover, they act as bi-directional synaptic organisers by mediating trans-synaptic interactions with presynaptic neurexins via cerebelin proteins. Although crystal structures of isolated domains of GluD receptors have been determined, structural insights into full-length receptors have been lacking, limiting further progress in the field. This work reports the structure of a full- length GluD1 receptor using single-particle cryo- EM data collected at beamline CM01.

Surprisingly, the GluD1 structures reveal a distinct architecture and domain arrangement in the receptor tetramer compared to other families of iGluRs (Figure 42). Unlike AMPA, Kainate and NMDA receptors, where the dimer partners at the amino-terminal domain (ATD) and ligand- binding domain (LBD) layer are swapped between the distal and proximal subunits of the receptor tetramer [2], GluD receptors adopt a unique non-swapped architecture (Figure 43). The separation of the extracellular domains at the ATD has been reported previously for AMPA and NMDA receptors; however, the non-swapped architecture is unique to GluD1. Structure-guided, cysteine-mediated, cross-linking experiments confirmed that the observed architecture was physiological. In addition, electrophysiology- based functional assays were used to show that the construct design does not affect receptor assembly and expresses functional receptors.

The unusual domain arrangement in GluD1 provides unrestricted freedom for the movement of the two extracellular domain arms in the detergent-solubilised receptor.

Fig. 42: The architecture and domain organisation of GluD1 receptors. a) Representative 2D class averages highlighting the conformational heterogeneity of the extracellular domains of GluD1. Cryo-EM density maps of (b) compact and (c) splayed GluD1 conformations fitted with atomic coordinates and showing the broadest face of receptor and 90°

rotated view. The four subunits A, B, C, D are coloured in orange, green, yellow and cyan, respectively.