Structural basis of the activation of metabotropic glutamate receptor 3

Dear Editor, Glutamate is used by most synapses in the brain and responsible for fast excitatory transmission, thus playing impor- tant roles in excitotoxicity and ammonium detoxi ﬁ cation in the brain. 1 Eight G protein-coupled metabotropic glutamate recep-tors (mGlus) are essential in sensing glutamate concentrations from the ten nanomolar to ten millimolar range in the brain. 2 The mGlu3 is in high sequence homology with mGlu2, but mGlu3 is of greater interest because it is responsible for the detection of very low concentrations of glutamate. 2 The mGlu3 is found in astrocytes and in both pre- and post-synaptic elements in neurons, whereas mGlu2 is mainly distributed in neurons, particularly in the preterminal region of axons, far from the active zone of neurotransmitter release. 3 Accumulating evidence supports a role of mGlu3 not only in maintaining synaptic homeostasis but also in promoting neuronal and astrocyte survival in several pathological conditions. 4 The mGlu3 has garnered attention as a potent therapeutic target for both psychiatric disorders and neurodegenerative diseases such as schizophrenia, Alzheimer ’ s disease, anxiety, depression, pain and addiction. 5,6 Polymorphic variants of the gene encoding mGlu3 are linked to schizophrenia. Furthermore, recent studies have suggested that negative allosteric modulators (NAMs) of both mGlu3 and mGlu2 induced rapid antidepressant-like effects through related but divergent mechanisms of action. 7 Moreover, the high sequence homology of mGlu3 and mGlu2 restricts the development of selective ligands, which demands the structures of mGlu3.Here,we present three cryo-EM structures of human mGlu3 homodimer: the agonist-bound state (bound with LY2794193),


Cryo-EM grid preparation and data collection
For the preparation of cryo-EM grids, 3 μl purified mGlu3 sample was applied onto a freshly plasma-cleaned holey carbon grid (GryoMatrix-M024, R1.2/1.3, 300 mesh, Au), blotted for 7 s at 100% humidity with a Vitrobot Mark IV (ThermoFisher Scientific) and plunge frozen into liquid ethane cooled by liquid nitrogen. The first dataset of agonist-mGlu3 was collected on a Titan Krios at 300 kV accelerating voltage in the Center of Cryo-Electron Microscopy, Zhejiang University (Hangzhou, China).
Micrographs were recorded using a Gatan K2 Summit direct electron detector in counting mode with magnification of 29,000×, corresponding to a pixel size of 1.014 Å. The total dose of 57.6 e-/Å2 was fractionated to 36 frames with 0.2 s per frame.
Nominal defocus values ranged from -1.5 to -1.8 μm. The second dataset of antagonist-NAM mGlu3 and the third dataset of antagonist-mGlu3 collected on a Titan Krios at 300 kV accelerating voltage in the Center of Cryo-Electron Microscopy, University of Science and Technology of China (Hefei, China). Micrographs were recorded using a Gatan K2 Summit direct electron detector in counting mode with magnification of 29,000×, corresponding to a pixel size of 1.01 Å. The total dose of 57.6 e-/Å2 was fractionated to 36 frames with 0.16 s per frame. Nominal defocus values ranged from -1.5 to -1.8 μm. The first dataset of agonist-mGlu3 included 11580 micrographs, the second dataset of NAM-bound inactive mGlu3 included 13106 micrographs and the third dataset of antagonist-mGlu3 included 4289 micrographs.

Imaging processing and 3D reconstruction
Three datasets were processed in cryoSPARC v2.15.0 1 . Movies were motion-corrected with built-in patch motion correction and contrast transfer function (CTF) estimation was performed with patch CTF estimation. Following CTF estimation, ~1000 particles were manually picked to generate 2D reference for auto-picking. For the first dataset of agonist-mGlu3, Auto-picking particles were extracted by four-times downscaling resulting in the pixel size of 4.056 Å. After three rounds of 2D classification, 862519 particles from well-defined 2D averages were extracted with a pixel size of 2.028 Å for further ab-initio reconstruction and heterogeneous refinement. A selected subset of particles from heterogeneous refinement were extracted with a pixel size of 1.014 Å and followed by C2 symmetry imposed in homogeneous refinement yielded a density map with nominal resolution of 3.82 Å according to the Fourier shell correlation (FSC) = 0.143 gold standard criterion. Followed heterogeneous refinement and Non-uniform refinement then improved the resolution to 3.68 Å. The second dataset of NAM-bound inactive mGlu3 was similarly processed in cryoSPARC. With a selected subset of particles yielded a 3.93 Å a density map from homogeneous refinement. Followed heterogeneous refinement and non-uniform refinement then improved the resolution to 3.71 Å. The third dataset of NAM-bound inactive mGlu3 was similarly processed in cryoSPARC. With a selected subset of particles yielded a 4.35 Å a density map from homogeneous refinement. Followed heterogeneous refinement and Non-uniform refinement then improved the resolution to 4.17 Å.

Model building
The initial model for the human mGlu3 receptor was derived from mGlu5 (PDB code: 6N51 and 6N52) followed by extensive remodeling using COOT 2 . The N-terminal residues 1-30, residues 118-140, ICL2 residues 669-687 and C-terminal residues 824-879 of mGlu3 were not built due to the lack of corresponding densities. Structure refinement and model validation were performed using phenix.real_space_refine module in PHENIX 3 . The final model was subjected to refinement and validation in PHENIX. Figures were prepared using UCSF Chimera 4 or UCSF Chimera X 5 .

CAMYEL biosensor assay for cAMP
The cAMP accumulation was measured by using the CAMYEL biosensor as previously

Statistical analysis
Statistical analyses were performed on at least three individual data sets analyzed by Graphpad prism. Data are means ± SEM from at least three independent experiments performed in technical triplicate. For dose-response experiments, data were normalized and analyzed using nonlinear curve fitting for the log (agonist) versus response (three parameters) curves.