Targeting mGlyR with nanobodies for depression

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: major_revision

Abstract
Development of therapies for neuropsychiatric conditions is one of the greatest challenges of modern medicine. Common limitations of traditional small molecule drugs include poor efficacy, off-target side effects and difficult druggability of many targets. In this study, we report a different approach deploying small engineered single domain antibodies, known as nanobodies, for the treatment of depression, a prevalent neuropsychiatric condition. We develop highly selective nanobodies for a recently discovered glycine receptor mGlyR crucially linked to pathophysiology of depression. Using a mouse model of stress-induced depression, we show that non-invasive intranasal delivery of nanobody produces rapid and lasting anti-depressant effect. We solve anatomic structure of mGlyR bound to nanobody and use a variety of cell-based approaches to reveal the mechanism of mGlyR modulation and its impact on neural circuitry. These findings support development of biologics for the treatment of intractable brain disorders.

Introduction
Major depressive disorder (MDD) is a prevalent neuropsychiatric condition that affects nearly 5% of the population in developed countries1. While there has been tremendous progress in treating depression, the efficacy of currently approved therapeutics is limited2. Exploring strategies and drug targets has been deemed a relevant goal for managing MDD3–5.

The molecular etiology of MDD is complex and not fully understood. Disbalance in neurotransmitter signaling is thought to trigger a range of maladaptive changes involving ion channels, kinases and second messengers, in particular cAMP, influencing synaptic communication and neuronal excitability6,7. These changes are precipitated by environmental factors such as stress, a major aggravating factor in MDD8. The neuronal circuits underlying the processing of emotional states and impacted by MDD are similarly complex and involve many structures, including the prefrontal cortex (PFC), a region heavily implicated in affective disorders and the effects of stress9.

MDD treatment has traditionally focused on the elements that mediate signaling by monoamine neurotransmitters targeted by a vast majority of currently approved antidepressants10. However, several recent medications also target GABA, glutamate and opioid receptors, highlighting the potential of other neurotransmitter systems in developing MDD treatments11,12. One such untapped system involves the neurotransmitter glycine. It is released by specific neurons and has distinct effects on neural circuits and the activity of neurons13,14. Glycine and its related naturally occurring compound taurine have been heavily implicated in mood regulation and depression15–17.

The effects of glycine had been thought to be mediated mainly by dedicated glycine receptor GlyR, an inhibitory ion channel18. Recently, a metabotropic receptor for glycine was discovered – mGlyR19. Formerly known as an orphan receptor GPR158, it exerts excitatory effects on neurons via modulation of the second messenger cAMP. The mGlyR is prominently expressed in the PFC, and its expression is regulated by stress20,21. The levels of mGlyR are markedly upregulated in patients diagnosed with MDD, and its knockout in mice produces anti-depressant phenotype and stress resilience20. The mGlyR employs an extracellular Cache domain for glycine recognition19. The binding of glycine or taurine to the ligand-binding pocket in this domain changes the activity of the associated Regulator of G Protein Signaling (RGS) complex at the intracellular side. This influences G protein signaling to second messengers and ion channels, thereby eliciting cellular response19. Notably, loss of RGS regulation also produces anti-depressant effects in mouse models22. Together, these observations point to mGlyR as an attractive target for developing anti-depressant therapies.

In this study, we explore the therapeutic relevance of targeting mGlyR as an antidepressant treatment. We develop a specific nanobody (Nb20) targeting the ligand-binding Cache domain of mGlyR, which mimics the effects of glycine. Through a series of studies, we show that Nb20 alters mGlyR function and its effects on neural circuits producing powerful antidepressant effects in mouse models when delivered in a non-invasive fashion.

Results
Development and characterization of nanobodies targeting mGlyR
Given the complete lack of selective chemical probes for mGlyR, we sought to obtain small protein ligands to alter mGlyR activity. The presence of extensive extracellular elements in mGlyR makes such a strategy attractive, considering recent success with other class C GPCRs23,24. We chose to generate single-domain antibodies (nanobodies) for their high affinity towards targets and emerging potential for therapeutic translation. Phage library prepared from a llama immunized with recombinant mGlyR was screened using HEK293 cell membranes containing mGlyR (Fig. 1a). After three rounds of enrichment and rescreening, 61 individual clones were chosen. Corresponding nanobodies were isolated following expression in E. coli and tested for mGlyR binding using a flow cytometry strategy (Fig. 1b). Three clones showed positive interaction with mGlyR-expressing cells (Supplementary Fig. 1). Of these, clone number 20 (Nb20) showed the most robust signal and was chosen for further studies.

First, we have used flow cytometry to characterize Nb20 binding to mGlyR. Cells transfected with Venus-tagged full length mGlyR were incubated with purified myc-tagged Nb20 and its interaction with cells was monitored by APC-conjugated antibodies against myc (Fig. 1b). Using this approach, we were able to detect robust labeling of majority of mGlyR-transfected cells with Nb20 when excess Nb20 was used in the assay (Fig. 1c). Control experiments with cells lacking mGlyR, Nb20 or both showed no labeling of cells with Nb20 (Fig. 1c, Supplementary Fig. 2a). Titration experiments with increasing concentrations of Nb20 showed saturable profile with EC50 of ~10 nM (Fig. 1d, e). The addition of mGlyR ligand glycine did not affect the binding of Nb20 with mGlyR (Supplementary Fig. 3). This interaction was specific to mGlyR, as we observed no Nb binding with other GPCRs, including related GPR179 and unrelated D1R (Supplementary Fig. 2b).

Next, we characterized the binding using surface plasmon resonance (SPR). In these experiments, we purified the recombinantly expressed extracellular portion of mGlyR (Ecto-mGlyR) tagged with an Fc tag and immobilized on the SPR chip via a mouse anti-human IgG CH2 monoclonal antibody (Fig. 1f). Applying increasing concentrations of Nb20 showed robust binding to the immobilized Ecto-mGlyR with an estimated K = 375 nM (χ2 = 1.07; K = 4.19 × 104 ms−1 and a K = 1.57 × 10−2 s−1 (Fig. 1g). The higher observed affinity of Nb20 in the cell-based applications, as compared to experiments with purified recombinant proteins, may be caused by an avidity effect that effectively enhances the binding to the low nanomolar level. Together, these data support the development of a selective affinity tool for mGlyR-Nb20.

Nb20 inhibits mGlyR signaling via RGS7/Gβ5 complex
To study the functional consequences of mGlyR interaction with Nb20, we analyzed the ability of mGlyR to modulate the activity of the GTPase-activating Protein (GAP) complex RGS7/Gβ5, through which mGlyR transduces its signals. To monitor GAP activity of the mGlyR complex, we studied its impact on the kinetics of G protein deactivation using a Bioluminescence Resonance Energy Transfer (BRET) assay (Fig. 2a). This assay monitors changes in BRET signal upon interaction of Venus-Gβγ subunits with the masGRK3CT-Nluc reporter. Upon Gα deactivation, Venus-Gβγ dissociates from masGRK3CT-Nluc reporter, quenching the signal. As previously reported19, we found that the introduction of RGS7/Gβ5 accelerated deactivation of its substrate, Gαo (Fig. 2b, d). Application of Nb20 had no significant effect on either baseline Gαo deactivation or RGS7/Gβ5-assisted process (Fig. 2b, d). However, when mGlyR was co-expressed together with RGS7/Gβ5, Nb20 significantly decelerated Gαo deactivation (Fig. 2c, d), suggesting that it specifically inhibited the GAP activity of RGS7/Gβ5 through mGlyR. Dose-response studies showed that the IC50 of Nb20 on mGlyR is ~6 nM (Fig. 2e). In summary, these studies show that Nb20 serves as a selective inhibitor of GAP activity mediated by the mGlyR complex.

Structural basis of mGlyR regulation by Nb20
To gain insights into the mechanisms underlying Nb20-mediated modulation of mGlyR, we obtained a high-resolution structure of mGlyR in complex with Nb20 using cryogenic electron microscopy (CryoEM) both with and without RGS7-Gβ5 complex (Fig. 3a, b). Three-dimensional classification (3D) of the particles revealed two dominant 3D classes, Nb-20-mGlyR and Nb-20-mGlyR-RGS7/Gβ5. Subsequently, these classes were refined to resolutions of 3.49 Å and 3.89 Å, respectively, without applying any symmetry (Supplementary Fig. 4, Supplementary Fig. 5). To improve the map, local refinements using a soft mask corresponding to ECD-Nb20 and TM-RGS7/Gβ5 were performed. The quality of the obtained maps exhibited higher resolution features in the TM region but relatively low resolution in the ECD and Nb20 binding regions. Nevertheless, these maps allowed us to construct the complete models of the mGlyR-Nb20 and mGlyR-RGS complexes, guided by known structures of mGlyR and the nanobodies (Supplementary Fig. 6).

We found that Nb20 was bound to the lateral side of the dimeric interface formed by two ligand binding Cache domains of the mGlyR dimer (Fig. 3c). The binding interface is predominantly mediated by the complementarity-determining regions 1 (CDR1) and possibly 2 (CDR2) of Nb20, establishing extensive contacts with the α2 helix and the loop between the β2 and β3 strands (residues 195–200) of subunit A, as well as a loop between the α1 and α2 helices (residues 140–153) of subunit B of mGlyR (Fig. 3d). The CDR2 of Nb20 interacts with a groove located at the dimeric interface between the two cache domains of the mGlyR receptor. Within the CDR2, the residue W58 lies in close spatial proximity to W162 from the α2 helix of subunit A of mGlyR, suggesting a potential interaction between these residues. Additionally, polar contacts may occur between R57 of Nb20 and E166 of subunit A, as well as between the backbone carbonyl of T59 Nb20 and N143 of the neighboring subunit of mGlyR (Fig. 3d). However, given the relatively low local resolution of the Nb20 and ECD regions, precise placement of side chains was not possible, and therefore these potential interactions should be interpreted with caution. We validated the binding mode of Nb20 by mutating and replacing all 6 critical residues in CDR1 and 9 residues in CDR2 regions with alanine and glycine (see Materials and “Methods”). This resulted in a complete loss of interaction of mutant Nb20 (Nb20*) with mGlyR (Supplementary Fig. 7). However, we can not exclude the possibility that these mutations may also affect the nanobody more globally. Notably, the ligand binding pocket is located adjacent to the binding groove of Nb20 (Supplementary Fig. 8a, b).

To determine whether binding to Nb20 indeed induces conformational changes in mGlyR, we first compared the Nb20-mGlyR structure to the apo structure of mGlyR alone. Global untethered superimposition of mGlyR-Nb20 on mGlyR-apo revealed conformational changes across various domains, including the Cache, stalk, and transmembrane (TM) domains (Supplementary Fig. 8c–e). Notably, the stalk domain exhibited pronounced movements, with deviations of up to 4.2 Å (Supplementary Fig. 8d). We next compared the transmembrane (TM) region in Nb20-bound complex structure with that of the two previously determined structures of mGlyR–RGS7–Gβ5 without Nb20 (PDBs: 7SHF and 7EWP). While the overall TM architecture is conserved across structures, Nb20-structure showed notable deviations in TM3–TM4 and the connecting loop, ranging from relatively minor ~1.1 Å (7EWP) to substantial ~5.5 Å (7SHF) (Supplementary Fig. 8f, h).

To pinpoint the precise conformational changes in the extracellular domain induced by Nb20 binding, we aligned the transmembrane (TM) domains (Fig. 3e). Anchoring the TM that way allowed us to observe dramatic conformational changes in the extracellular domain (ECD) upon Nb20 binding to the receptor. Specifically, in the Nb20-bound structure, the ECD translated by as much as 12 Å and rotated by approximately 7° in relation to the mGlyR-apo structure (Fig. 3f). These observations suggest that binding to Nb20 starts the wave of events that eventually remodel the cytoplasmic interface of the receptor involved in interaction with RGS7/Gβ5 and transduction of the signal. To test this model, we further compared the cryoEM structure of Nb20-mGlyR-RGS7-Gβ5 with previously solved mGlyR-RGS7/Gβ5 structures (PDB: 7SHF, 7EWP) (Fig. 3g, Supplementary Fig. 8f, h). In the mGlyR-RGS7/Gβ5 structures, the ECD domain was highly flexible, resulting in low-resolution features as observed in 7EWP25 or scattered density at relatively higher resolution in 7SHF26 structures. The binding of Nb20 appeared to improve ECD stability in the presence of the bound RGS7-Gβ5 module, yielding a relatively well-resolved density for ECD. Strikingly, comparison revealed that the density corresponding to the RGS domain, which typically wraps around Gβ5 from below, was poorly resolved in the Nb20-bound structure, suggesting that the RGS domain adopts a more flexible conformation compared to mGlyR-RGS7/Gβ5 structures, where this region was resolved at higher resolution. (Fig. 3b, Supplementary Fig. 8f, h). Even after local refinement and analysis at various contour levels, the RGS domain density remained poorly resolved, suggesting that this domain becomes highly flexible upon binding the Nb20 to ECD. Superimposition of Nb20-bound and free structures by the 7TM regions suggested plausible changes. The DEP/DEX domain of RGS7, which directly contacts the receptor, adopted a distinct conformation in the Nb20-bound structure. Notably, the β3-hairpin loop becomes unresolved (Fig. 3g), which may affect the RGS complex. Nb20 binding appeared to shift CTH2 by 2.2 Å, induce a 1.2 Å displacement in RGS7, and 1.5 Å movement in the TM3-TM4 connecting loop, and up to a 5.5 Å (when compared with 7SHF) changes in the TM3, which serve as the contact site for RGS7 (Supplementary Fig. 8f, l). However, the exact side-chain orientation at these regions could not be resolved due to the low local resolution, limiting the structural analysis to the level of secondary structure elements. A high-resolution structure of RGS7-Gβ5 will be required to delineate the remodeling of hydrogen bonding and hydrophobic interactions following Nb20 binding. Although the Gβ5 in the Nb20-bound structure appears to shift by approximately 5 Å compared to its position in the structure without Nb20, this displacement should be interpreted cautiously, given the poorly resolved density for Gβ5 (Fig. 3g, Supplementary Fig. 8g). Together, structural studies suggest a model where binding of Nb20 to the extracellular ligand binding Cache domain of mGlyR may promote specific stabilized conformation of the receptor which possibly remodels its intracellular interaction with the RGS7/Gβ5, leading to a higher flexibility of its catalytic RGS domain, decoupling it from regulating Gα proteins, e.g. by changing its orientation towards the membrane where Gα protein are residing.

Nb20 produces anti-depressant effects in mice
Our functional data indicate that Nb20 blocks the ability of mGlyR-RGS7/Gβ5 complex to regulate G protein signaling. Previous studies indicated that genetic deletion of either mGlyR20 or RGS722 produced a substantial antidepressant phenotype in mice and stress resilience. Therefore, we have next tested whether administration of Nb20 in vivo would have similar behavioral effects. Sequence analysis of llama-derived Nb20 reveals favorable immunogenicity profile likely making it suitable for in vivo applications in other species (Supplementary Fig. 9). Mice were injected, delivering either Nb20 or vehicle control into the brain (Fig. 4a) and evaluated in a panel of behavioral tests that generally assess various aspects of affective-like states 24 h later (Fig. 4b). We found that mice that received Nb20 buried fewer marbles in the marble burying test, had reduced immobility in the tail suspension and forced swim tests relative to control animals but their behavior in the elevated plus maze did not reach threshold for statistical significance when compared to untreated mice (Fig. 4b). Calculating overall emotionality score based on multiple measures confirmed that mice treated with Nb20 displayed prominent antidepressant-like phenotype (Fig. 4c). Remarkably, these behavioral differences between the groups persisted for at least two weeks following the treatment at which point anxiolytic effect slightly increased (Supplementary Fig. 10). To confirm the specificity of the effects, we used mutated Nb20* incapable of binding to mGlyR and found that treatment of mice with these nanobodies did not produce significant effects in any of the paradigms used (Supplementary Fig. 11). We further conducted a panel of tests to measure the effect of Nb20 extending the range of behavioral testing to better understand Nb20 actions. We found that treatment of mice with Nb20, but not inactive Nb20*, alleviated novelty-induced hypophagia, an approach behavior influenced by depression/anxiety-related affective states (Supplementary Fig. 12a). Interestingly, Nb20 further enhanced an ability of mice to recognize novel object suggestive of the positive effect in the cognitive domain (Supplementary Fig. 12b). Importantly, Nb20 treatment had no effect on locomotor activity (Supplementary Fig. 13a) or motor performance (Supplementary Fig. 13b) of mice.

To further explore the therapeutic utility of Nb20, we assessed its effects in a stress-induced depression model where mice are exposed to a chronic variable stress (Fig. 4d, Supplementary Fig. 14). In this study, Nb20 was also administered non-invasively through intranasal delivery, given the translational relevance of this method for treating depression27–29. Furthermore, the effects were benchmarked to the effects of rapid anti-depressant treatment with ketamine delivered the same way (Fig. 4d, e). Remarkably, we found that intranasal delivery of Nb20 produced a rapid and powerful antidepressant effect in stressed mice (Fig. 4F) with similar effects in males and females (Supplementary Fig. 15). The extent of this effect matched the antidepressant effects of ketamine across all behavioral paradigms. We did not detect the effect of Nb20 on either anxiety-related behaviors of stress-exposed mice or general motor activity (Supplementary Fig. 16). Neither did we observe Nb20 influencing the hedonic component in the sucrose preference task (Supplementary Fig. 16). Furthermore, Nb20 was able to normalize stress-induced elevation of corticosterone, prominent in female mice (Supplementary Fig. 17). Together, these results indicate that Nb20 produces antidepressant-like effects in a mouse model of stress-induced depression.

Inhibition of mGlyR with Nb20 modulates physiological properties of mPFC neurons
We finally sought to assess the impact of Nb20 on the activity of ex vivo intact neural networks by examining the intrinsic properties of layer II-III neurons in the prelimbic cortex, where mGlyR is prominently expressed29. Knockout of mGlyR or its inhibition with endogenous ligand glycine has been shown to increase the excitability of pyramidal neurons in layer II-III28, among other effects on synaptic physiology, which vary across different brain regions and neuronal populations30–32. Thus, blockade of mGlyR with Nb20 would be predicted to increase the excitability of layer II-III neurons. To test this hypothesis, we incubated brain slices with Nb20 for 10 min prior to recordings and compared excitability of layer II-III neurons with the untreated slices or slices incubated with control Nb20* nanobody deficient in mGlyR binding (Fig. 5a). Pre-incubation with Nb20 significantly increased the number of action potentials fired over the ramp while decreasing the amount of current necessary to elicit the first action potential (Fig. 5b–d) without changes in the resting membrane potential (Fig. 5e, Supplementary Fig. 18). In contrast, control Nb20* had no effect on neuronal firing, rheobase current or resting membrane potential (Fig. 5b–d, Supplementary Fig. 18). Together, these results indicate that inhibition of mGlyR with Nb20 increases excitability of layer II-III neurons in prelimbic cortex, in a manner similar to genetic loss of mGlyR or its inhibition by glycine.

Methods
Animals
All animal experiments were approved by the Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology Institutional Animal Care and Use Committee (IACUC) as well as by the Comité d’Ethique en Expérimentation animale Val de Loire (C2EA-19; APAFIS #46226-2022022509329925 v6). All experimental procedures were conducted in accordance with guidelines by NIH and the European Communities Council Directive 2010/63/EU. Animal studies are reported in compliance with the ARRIVE guidelines. The Gpr158−/− mice were purchased from KOMP (Gpr158tm1(KOMP)Vlcg) and maintained on a C57/Bl6 background and bred as heterozygous pairs to generate Gpr158−/− and Gpr158+/+ littermates. After weaning, male and females were separately group-housed under standard conditions in a pathogen-free facility on a 12:12 light:dark hour cycle with access to food and water ad libitum. For the chronic variable stress paradigm, wild-type male and female adult (8-week-old) C57Bl/6J mice (Charles River, Massachusetts) were habituated for one week before experimental manipulation. These mice were single-housed at room temperature (~24°C) under a 12-h light/dark cycle (07:00-19:00) with ad libitum access to water and food, except during testing.

cDNA constructs
Dopamine D2 receptor (cDNA Resource Center: #DRD0200001), RGS7 (GenBank: AY587875), Gβ5 (GenBank: NM_016194), GαoA (cDNA Resource Center: #GNA0OA0000) in pcDNA3.1(+) were purchased from cDNA Resource Center (https://www.cdna.org). masGRK3ct-Nluc, Venus-156-239-Gβ1, Venus-1-155-Gγ2 were synthesized by GenScript in pcDNA3.1+. pCMV5 plasmid encoding GαoA was a gift from H. Itoh (Nara Institute of Science and Technology, Japan). GPR158 ectodomain (aa 1-417) was subcloned into a previously described Fc and 6xHis tagged vector32. Nanobody production was done using the pCANTAB vector (phage display) and promising candidates, including Nb20, were subcloned into pET28a. The complete sequence of Nb20 is as follows: MAEVQLQESGGGLVQAGGSLRLSCAASGSIGNIYIMGWYRQTPGPQRELVATIRTVRWTKYEDYADSVKGRFTISDDDAKNTVYLQMNSLKPEDTAVYYCNYKDYNAPSDGYWGQGTQVTVSSEPKTPKPQ. To generate control non-binding nanobody (Nb20*), Nb20 cDNA was mutated to replace 30IGNIYI35 sequence in CDR1 with 30GGAGAG35 sequence and 54RTVRWTKYE62 sequence in CDR2 with 54GAVGGAAAG62 sequence using direct mutagenesis by PCR with 2 sets of primers: CDR1 Forward primer: GCGGCGGTGCTGGCGCTGGCATGGGCTGGTACCGCCAG; CDR1 Reverse primer: CGCCAGCACCGCCGCTTCCAGAGGCTGCACAGGAGAGTC and CDR2 Forward primer: CAGCCGCAGCCGCAGCCGCAGCCGACTATGCAGACTCCGTAAAGGGC; CDR2 Reverse primer: CTGCGGCTGCGGCTGCGGCTGCAGTTGCGACCAGCTCGCG.

Chemical and drugs
The following chemicals were used. Dopamine hydrochloride (MilliporeSigma Cat# H8502), Haloperidol (MilliporeSigma Cat# H1512), Dulbecco’s Phosphate-Buffered Saline (PBS) (Gibco Cat# 10010-023) with 0.5 mM MgCl2 and 0.1% glucose, glycine (National Diagnostics # EC-405), Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific Cat# 11965-092), Fetal bovine serum (Genesee Scientific Cat# 25-550), Sodium pyruvate (Thermo Fisher Scientific Cat# 11360-070), MEM non-essential amino acids (Thermo Fisher Scientific Cat# 11140-072), Penicillin-streptomycin (Thermo Fisher Scientific Cat# 15140-122), Matrigel (Corning Cat# 356230), METAFECTENE® PRO (RKP203/RK092820, Biontex Germany), Dulbecco’s phosphate-buffered saline (MilliporeSigma Cat# D5652), PEG 6000/2M NaCl (Teknova # P4168), Nano-Glo luciferase (N113B Promega), Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich # I6758), Kanamycin (Thermo Fisher Scientific # 11815032). The following chemicals were prepared: LEW (50 mM NaH2PO4, 300 mM NaCl, pH adjusted to 8.0 using NaOH), 2xYT (16.0 g/l tryptone, 10 g/l yeast extract, 5 g/l sodium chloride, Final pH 6.8 ± 0.2 at 25°C, autoclaved).

Llama immunization, phage display
One llama was immunized in strict accordance with good animal practices following the EU animal welfare legislation law with HEK293 cell membrane expressing mGlyR at Eurogentec (Belgium). Blood samples from immunized llama were harvested 87 days after immunization, and RNA from leukocytes was extracted using LeukoLOCK Total RNA Isolation system (Life Technologies # AM1923) accordingly to manufacturer instructions. Reverse transcription was performed on extracted RNA, and cDNA was amplified by PCR using 8 pairs of primers designed to amplify the variable heavy only domains (VHH) of non-conventional IgG2 and IgG3 and introduce NotI and SfiI cleavage sites, respectively (in bold in the following sequence).

Forward primers:
VH_11: GTCGTCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTAGTCTGGGGGAGG
VH_12: GTCGTCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGCAGGMGTCTGGGGGAGG
VH_14: GTCGTCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGCAGGCGTCTGG
VH_13: GTCGTCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGCAGGAGTCWGG

Reverse primers
VH_sh: GCTGCTGCGGCCGCGGGGTCTTCGCTGTGGTGCGC
VH_lg: GCTGCTGCGGCCGCTTGTGGTTTTGGTGTCTTGGG

Amplified cDNA was purified and digested with NotI and SfiI, following by ligation into the phagemid vector pCANTAB5 and electroporation of competent E. coli TG1 cells. Bacteria were grown in 2xYT broth containing 2% glucose at 37°C under agitation until OD 600nm reached 0.5. Subsequently, helper phage KM13 (2×1011 units) was added for 30 min at 37°C without agitation. Infected bacteria were pelleted by centrifugation at 4000g for 20 min at 4°C, and the pellet was resuspended in 2xYT containing 120 µg/ml ampicillin and 50 µg/ml kanamycin. The phage library was grown overnight at 37°C with agitation. Phages expressing a nanobody library were harvested and purified by centrifugation at 8000g for 15 min at 4°C, supernatant was precipitated using 20% of PEG 6000/2.5 M NaCl for 1 h at 4°C, followed by centrifugation at 8000g for 15 min at 4°C and pellet was resuspended in PBS-glycerol 15% and stored at +4°C.

Nanobody identification via phage library screening
To obtain anti-mGlyR-specific nanobody clones, two steps of depletion were first performed. Library containing 1012 phage units was incubated in the first well of a MaxiSorp plate (Thermo Fisher Scientific # 441653) coated with 100 µg of HEK293 cell membranes mock-transfected for 1 h at RT, and supernatant was transferred to a second identically coated well for 1 h at RT in order to reduce non-specific binders. A last step of selection was performed using the remaining phages incubated on one well coated with 100 µg of cell membranes expressing mGlyR for 2 h at RT. After washing, bound phages were eluted with Tris 50 mM pH = 8, 1 mM CaCl2, Trypsine 1 µg/ml for 15 min at RT. Eluted phages were recovered and amplified with E. coli TG1 cells infected in 2xYT broth containing ampicillin and 2% glucose overnight at 30°C with agitation. Next day, 2×109 units of helper phage KM13 (AKA VCSM13 Agilent Technologies # 2002521) were added to the amplified TG1 cells for 1 h at 37°C, followed by centrifugation at 3000g for 10 min at RT. Pellet was resuspended in 2xYT broth containing 100 µg/ml ampicillin and 25 µg/ml kanamycin and incubated overnight at 30°C with agitation. To separate TG1 cells and amplified phages, the overnight culture was centrifuged at 3000g for 30 min at 4°C and phages in the supernatant were precipitated for 30 min on ice using 20% PEG-6000/NaCl 2.5 M, followed by a centrifugation at 10,000g for 10 min at 4°C. Pellet was resuspended in PBS with 15% glycerol and stored at −20°C, and the rest was used in the second round of panning selection as described previously. A total of 3 rounds of panning were performed to identify 61 individual clones. Each clone was produced directly from individual TG1 colonies induced by isopropyl-β-26-D-thiogalactopyranoside (IPTG) for flow cytometry screening.

Protein production and purification
Individual nanobody cDNAs were fused with a c-myc and 8xHis tags at the C-terminus, subcloned into the pET28a vector using the in-fusion HD cloning kit (Takara Bio # 102518) and sequenced. Freshly transformed E. coli BL21 DE3 (NEB # C2527H) with pET28a plasmid encoding nanobody was grown at 37°C, shaking at 220 rpm in Terrific Broth (Kd Medical Inc # 501018968) containing 50 µg/ml kanamycin until OD 280nm = [0.6–0.8]. Protein expression was then induced by 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), and bacteria were grown overnight at 28°C with shaking at 220 rpm. Bacteria were collected, centrifuged, and the pellet was lysed by sonication in LEW 1X at 4°C. Periplasmic solution were harvested after centrifugation at 17,000g, 30 min, 4°C and purified on a Ni-IDA column (Macherey Nagel # 745160) according to manufacturer recommendations. Ecto-GPR158-Fc was produced in HEK293FT cells (supernatant) purified (Nickel column) dialyzed/concentrated (Amicon tube 30 kDa). Protein concentration in supernatant was determined using UV spectroscopy (Nanodrop).

Cryo-EM sample preparation and data acquisition
The cryo-EM sample was prepared as described previously26. The protein sample, the purified Nb20-mGlyR-RGS7-Gβ5 complex, was prepared for cryo-electron microscopy (Cryo-EM) imaging. A total of 3.0 µL of the protein sample was applied to glow-discharged 200 mesh gold grids (Ultra Aufoil R1.2/1.3) inside an FEI Vitrobot Mark IV (Thermo Fischer Scientific). The Vitrobot was maintained at 4°C with 100% humidity. Prior to blotting, a blot force of 2, a blot time of 2 s, and a wait time of 20 s were applied to remove excess sample. The grids were then plunge-frozen in liquid ethane to preserve their vitrified state.

Cryo-EM imaging of the Nb20-mGlyR-RGS7-Gβ5 protein complex was performed using a 300 kV Titan Krios electron microscope equipped with a Gatan K3 Summit direct electron detection (DED) camera (Gatan, Pleasanton, CA, USA) and a post-column GIF Quantum energy filter operating in counting mode. The microscope was calibrated to a magnification of 105,000, resulting in a nominal pixel size of 0.873 Å. A total of 6689 movies were collected, covering a defocus range of −1.5 to −2.0 µm. The total dose applied was 40 e^−/Å^2, achieved by using a dose rate of approximately 12.5 e^-/s/phys pixel per frame across 40 frames, resulting in a total exposure time of 2.5 s.

Image processing and 3D reconstruction
The mGlyR cryo-EM dataset was processed using56 and cryoSPARC57. The 6689 movies were initially motion-corrected for beam-induced motion using MotionCor258 within RELION. Motion corrected images were then imported into cryoSPARC57, where the patch Contrast transfer function (CTF) estimation tool was used for CTF estimation.

For particle picking, the TOPAZ algorithm, which employs a convolutional neural network algorithm implemented in CryoSparc, was used59. A training set of 1000 micrographs were used to generate a trained model, which was subsequently used for particle picking across the entire dataset, resulting in a total of 3,247,619 particles, which were then extracted from the micrographs with a box size of 340. The extracted particles underwent three rounds of reference-free 2D classification to discard poor-quality particles. The protein particles exhibiting favorable 2D class averages were combined and subjected to several rounds of ab initio and heterogeneous refinement. This iterative refinement process led to the generation of two subsets one containing 91,859 particles corresponding to the Nb20-mGlyR-RGS complex, and the other consisting of 121,523 particles corresponding to the mGlyR-Nb20 complex. The final resolution of these subsets was estimated to be approximately 3.49 Å and 3.89 Å, respectively, using the gold standard Fourier shell correlation (GSFSC) method within cryoSPARC57. To improve the maps for ECD-Nb20 and TM-RGS7-Gβ5, focused refinement was performed using soft masks (0.001 threshold, 12-pixel padding, 3-pixel dilation) corresponding to each region. This focused refinement approach produced maps at 4.14 Å for ECD-Nb20 and 4.18 Å for TM-RGS7-Gβ5, enabling model building. EM density visualization was done in UCSF Chimera60.

Model building and refinement
The models of mGlyR-Nb20 and Nb20-mGlyR-RGS7-Gβ5 were built based on the previously determined structure of mGlyR (PDB ID: 7SHE and 7SHF) using COOT61. When building the model for mGlyR, the density corresponding to Nb20 was observed, indicating the presence of the bound nanobody. The shape and size of this density were consistent with the bound nanobody. The Nb20 model generated using the AlphaFold method was docked into the cryo-EM map and manually adjusted, built, and refined using COOT61.

The cryo-EM map of Nb20-mGlyR-RGS7-Gβ5 had a final resolution of 3.89 Å. The map exhibited well-resolved density for the transmembrane (TM) domain but relatively low density for RGS7, with the RGS domain missing from the maps. To build the atomic model of the Nb20-mGlyR-RGS7-Gβ5 structure, the mGlyR-Nb20 model generated in this study (from the same dataset) was used as a template. The model was docked into the map using UCSF Chimera and further manually adjusted, built, and refined in COOT.

In the Nb20-mGlyR-RGS7-Gβ5 map, the density for the RGS domain of RGS7 was poorly resolved and fragmented, making it challenging to build a reliable model. The TM domain of both the mGlyR-Nb20 and Nb20-mGlyR-RGS7-Gβ5 models underwent iterative manual building in COOT, followed by real-space refinement in PHENIX62. The Gβ5 and RGS7 regions were not resolved well, and therefore, these regions were docked into the map as rigid bodies. The RGS domain, which is not visible in the map, was not built and remains absent from the final model. Local rotamer fitting and restrained group ADP refinement were also performed. The resulting models were refined against both the unfiltered half maps and sum maps in real space using PHENIX. Structures were visualized, and figures were prepared in UCSF Chimera60, ChimeraX63,64 and PyMOL65. The data collection and refinement statistics are listed in Table 1.

Culture and transfection of mammalian cell lines
HEK293FT cells were obtained from Thermo Fisher and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (v/v), minimum Eagle’s medium non-essential amino acids, 1 mM sodium pyruvate, and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin) at 37°C in a humidified incubator containing 5% CO2. Cells were transiently transfected using Metafectene Pro (Biontex, Germany) in 96 96-well plate following the manufacturer’s instructions.

Cell-based bioluminescence resonance energy transfer (BRET) assays
HEK293FT cells were seeded in a white flat-bottom 96-well plate (Greiner Bio-One) at 50,000 cells per well and transiently transfected using Metafectene Pro with the manufacturer's instructions. pcDNA3.1 plasmids coding for Dopamine D2R (1), mGlyR (1), RGS7 (1), Gb5 (1), Gao (2), Venus-1-155-Gγ2 (1), Venus 156-239-Gβ1 (1) and masGRK3ct-Nluc (1) were used to transfect cells (ratio in parenthesis). An empty vector (pcDNA3.1(+)) was used in order to normalize the quantity of transfected DNA. 24 h after transfection in DMEM complete medium supplemented with 0.1% of Matrigel (Corning), cells were washed with BRET buffer (Dulbecco’s Phosphate-Buffered Saline (PBS) containing 0.5 mM MgCl2 and 0.1% glucose). Measurements of BRET between Venus-Gβ1γ2 and masGRK3ct-Nluc were performed to monitor the release of free Gβγ dimers after activation of heterotrimers containing Gα subunits in living cells, as described before66. Cells were incubated with 1 µM of Nb20 or with 100 µM of glycine or both when indicated and with the NanoLuc (Nluc) substrate at the manufacturer’s instructions. In order to release Gαo and start deactivation of Gαo, 100 µM of dopamine and 100 µM of haloperidol were injected sequentially and automatically (t=10s and t=25s) by the plate reader (PHERAstar FSX, BMG Labtech), and dual-luminescence was measured at 475±30 nm and 535±30 nm. The BRET signal was determined by calculating the ratio of the light emitted by the Venus-Gβγ (535 nm with a 30 nm band path width) over the light emitted by the masGRK3ct-Nluc (475 nm with a 30 nm band path width). The average baseline value (basal BRET ratio) recorded prior to agonist stimulation was subtracted from the experimental BRET signal values, and the resulting difference (netBRET ratio) was normalized against the maximal netBRET value recorded upon agonist stimulation. The rate constants (1/τ) of the deactivation phases were obtained by fitting a one-phase exponential decay curve to the traces with Graphpad Prism 9.0. The kGAP rate constants were determined by subtracting the basal deactivation rate (kapp) from the deactivation rate measured in the presence of exogenous RGS protein.

Flow cytometry
HEK293FT cells were cultured in 6-well plates at a density of 1.106 per well and transfected with 2 µg of cDNA of mGlyR or empty pcDNA3.1+ in control experiments, using Metafectene Pro. 48 h after transfection, cells were mechanically detached by pipetting up and down, washed in PBS supplemented with 0.1% BSA, counted and incubated in PBS-0.1% BSA for 1 h at 4°C under rotation. Nanobody-20 (Nb20) and 10 µl of anti-myc-APC conjugated antibody (R&d Systems # IC3696A) were added and incubated in the dark with rotation, at 4°C for 1 h. After 3 washes, cells were analyzed in the LSR-II BD flow cytometer. A gating strategy to sort individual cells from debris and doublets was used as indicated in each figure. Sorted cells were measured for fluorescence in respective channels. Negative control conditions were used to set the positive threshold for APC (mock cells incubated with Nb and anti-myc-APC antibody), and for Venus (mock transfected cells). Acquired data were analyzed using FlowJo software (FlowJo).

Surface plasmon resonance (SPR)
Surface plasmon resonance (SPR) measurements were performed on a Biacore X100 instrument at 25°C using 1x HBS-EP+ (Cytiva) as a running buffer. A mouse anti-human IgG CH2 monoclonal antibody (Cytiva) was immobilized to a density of ~9500 response units (RU) on a CM5 sensor chip via standard NHS/EDC coupling methods (Cytiva). Subsequently, GPR158-Fc at 10 μg/mL was captured to ~1,800 RU on the active flow cell. A concentration series with two-fold dilutions (500–31.25 nM) of the nanobody Nb20 were injected using a multi-cycle method. The lowest concentration (31.25 nM) was repeated to confirm regeneration of the sensor chip. Biacore X100 Control Software 2.0.1 (Cytiva) was used to collect data, and Biacore X100 Evaluation Software 2.0.1 (Cytiva) was used to analyze data.

Brain slice preparation and whole cell recordings
Electrophysiological recordings from layer II-III neurons of the prelimbic cortex were performed with mice of either sex aged between 4–12 weeks. Mice were anesthetized with isoflurane and decapitated. The brain was quickly removed and rested for 30 s in ice-cold oxygenated solution containing (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2·, 10 MgCl2, (adjusted to 7.3–7.4 pH with HCl). Coronal slices (300 μm thick) were cut on a vibratome (VT1200S, Leica), mounted on a porous membrane and incubated for 12 min at 34°C in NMDG. Slices were then transferred to a modified HEPES ACSF containing (in mM): 92 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 Na-ascorbate, 2 thiourea, 3 Na-pyruvate, (adjusted to 7.3–7.4 pH with NaOH) and allowed to recover for 1 h at room temperature. For recordings, slices were transferred to a submerged recording chamber where they were continuously perfused at 2 ml/min with oxygenated ACSF containing the following (in mM): 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 18 NaHCO3, 1.2 NaH2PO4, 10 glucose, in presence of the following synaptic blockers: picrotoxin (100 µM), strychnine (1 µM), CNQX (20 µM), APV (50 µM). Pipets (3–5 MΩ) were pulled from P-1000 (Sutter Instruments, CA) and filled with an intracellular solution containing the following (in mM): 119 K-MeSO4, 12 KCl, 1 MgCl2, 0.1 CaCl2, 10 HEPES, 1 EGTA, 0.4 Na-GTP, 2 Mg-ATP (280–300 mOsm, pH 7.3 adjusted with KOH). Slices were incubated with Nb-20, Nb-20* (1 µM), or ACSF and changes in neuronal excitability were assessed by counting the number of spikes evoked in response to a 1-s depolarizing ramp ranging from 0 to 200 pA with a 20-s intertrial interval. The Rheobase current was defined as the minimum current necessary to elicit the first AP. Acquisition was done using Clampex 10.7, MultiClamp 700B amplifier and Digidata 1440A (Molecular Devices, CA). Data were analyzed with Clampfit 10.7.

Mousestudies
No statistical method was used to predetermine sample size. No data were excluded from the analyses. No specific randomization methods were used. Animals were randomly assigned to experimental groups depending on genotype. Experimenters were blinded to the treatment groups.

Drug treatments. Intracerebroventricular (ICV) administration was performed according to a previously described method67. Mice were injected with 5 µl of purified and endotoxin-free nanobody solution in saline (1.92 mg/ml) of nanobody or 5 µl of vehicle. Behavioral evaluation was conducted starting 24 h after treatment over the course of three days. Marble burying was performed in the morning on day one, followed by the elevated plus maze in the evening. Tail suspension was done the next day, and the force swim test was performed on day three. The same sequence and timing of behavioral testing was repeated two weeks after treatment. For the intranasal (IN) administration, 24 h after the last stressor, mice received two treatments on the same day, separated by a 6-h interval. Each treatment involved the application of 10 µl of purified, endotoxin-free nanobody solution in saline (0.95 mg/ml) or racemic ketamine hydrochloride (20 mg/kg; VetaKet; Patterson Veterinary; Cat# 78925834) dissolved in saline, delivering 5 uL to each nostril, using a P-10 pipette.

Stress paradigm. The chronic variable stress (CVS) paradigm consists of daily exposure to one of three stressors over 21 consecutive days. All mice are exposed to one stressor per day, and the three stressors are repeated every three days. The times for each stressor vary from day to day to increase unpredictability. The stressors include restraint stress, where mice are placed in ventilated 50 mL conical tubes for 1 h in the home cage. The next stressor is foot shock, which is performed in shock boxes (6 mice can be simultaneously run). Mice receive 100 mild foot shocks at 0.4 mA for 1 h at random intervals. The final stressor is 30 min of predator odor exposure. 15 uL of TMT (Fisher Scientific; 501844430) is pipetted onto a cotton tip applicator and placed into a clean, empty standard mouse cage. The single housing is also considered an additional stressor on the mice. Termination criteria were used to determine whether mice should continue in the stress paradigm, including bleeding, excessive weight loss (>20% initial), and hunched or moribund phenotypes. Behavior was run 24 h after drug treatment in counterbalanced groups, and animals were numbered to maintain blinding in the manually scored tests. 48 h after the final behavioral test, mice were sacrificed.

Marble burying. Marble burying (MB) was conducted in a standard mouse cage (27×16.5×12.5 cm) with 5 cm corncob bedding and 20 glass marbles overlaid in a 4×5 equidistant arrangement. Background white noise (approximately 70 dB) was used during trials. The mouse was placed in the center of the cage, and testing consisted of a 30 min exploration period. The marbles, at least half-buried at the end of the trial, were counted as buried.

Sucrose splash test. The splash test (ST) was performed using a 10% sucrose solution freshly prepared the day of the test (Sigma-Aldrich; S9378) and was sprayed on the dorsal coat of the mouse (~0.35 mL per mouse), and placed into an empty, inescapable cylindrical Plexiglas container (121 cm in length and 15 cm in diameter). Mice were habituated to the cylinder for 5 min before spraying with sucrose solution and returning to the container. The behavior of the mouse was recorded for 5 min and later scored manually by blinded observers. The videos were scored for total grooming time. The scores of the observers are averaged to give final values.

Elevated plus maze. The elevated plus maze (EPM) was performed using a black, plexiglass elevated plus maze (apparatus with two open and two enclosed arms, 33×6 cm, with a wall of 25 cm on the closed arm, elevated 60 cm from the floor; Med Associates, St. Albans, VT). Lighting for the maze was set at 200 lux in the center of the plus maze, 270 lux on the open arms, and 120 lux on the closed arms. Background white noise (approximately 70 dB) was used during trials. Mice were placed in the center of the elevated plus maze and left to explore for 5 min in a dim light condition. Mice were recorded using Ethovision XT, and the time spent in the open and closed arms, as well as the number of entries from the closed to the open arm, were calculated.

Tail suspension test. The tails of the mice were wrapped with tape that covered approximately 4/5 of the tail length and then fixed upside down on a hook. The immobility time of each mouse was recorded and tracked over a 6 min period using Ethovision XT. Automated tracking values were validated via manual scoring by a blind observer.

Force swim test. The Porsolt Forced Swim Test (FST) was conducted using a vertical clear glass cylinder (10 cm in diameter, 25 cm in height) filled with water (25°C). The mice spent 6 min in the water, and immobility was scored. A mouse was regarded as immobile when floating motionless or making only those movements necessary to keep its head above the water. Automated tracking values were validated via manual scoring by a blind observer.

Analysis of emotionality score. The behavioral paradigm used to calculate the emotionality score were performed in the following order: MB, ST, EPM, TST and FST. To obtain a comprehensive measure for emotionality, we used z-scoring methodology to integrate standard measures of anxiety-like and depressive-like behaviors, as previously described68. The testing parameters analyzed were as follows: marble burying (number of marbles buried), elevated plus maze (time spent on open arm, number of entries into the open arm), tail suspension test (immobility) and forced swim test (immobility). For each parameter, the z-score for every individual animal was calculated using the previously described approach20. Briefly, for each parameter, the z-score of each individual animal was calculated using the formula Z = X − μ / σ, where X represents the individual data point, m represents the mean of the control group, and s represents the standard deviation of the control group. The emotionality score (ES) for each individual subject was first averaged within test, and then across each test to ensure equal weighting of all tests. ES = ZMB + ZEPM + ZTST + ZFST / number of tests. The mean emotionality score for each group is an average of the individual scores within each group for each experiment.

Novelty-suppressed feeding. Novelty-suppressed feeding (NSF) was measured in 24-h food-deprived mice, isolated in a standard housing cage for 30 min before individual testing. Three pellets of ordinary lab chow were placed on a white tissue in the center of each arena, lit at 80 lux. Each mouse was placed in a corner of an arena and allowed to explore for a maximum of 15 min. Latency to feed was measured as the time necessary to bite a food pellet. Immediately after an eating event, the mouse was transferred back to the home cage (free from cage-mates) and allowed to feed on lab chow for 5 min. Food consumption in the home cage was measured.

Novel object recognition. The experiments were conducted in 4 equal square arenas (50×50 cm) separated by 35 cm-high opaque gray Plexiglas walls. The light intensity of the room was set at 15 lux to facilitate exploration and minimize anxiety levels. The floor was a white Plexiglas platform (View Point, Lyon, France), spread with sawdust. The room was equipped with an overhead video camera connected to a computerized interface, allowing visualization and recording of behavioral sessions on a computer screen in the adjacent room. The experimental paradigm lasted for 2 days (3, 4). On day 1, the animals were placed in an arena for a 15 min-habituation session with two copies of an unfamiliar object (T-shaped plastic tubing, 1.5×3.5 cm). These objects were not used later for the recognition test. On day 2, the recognition test was performed, consisting of 3 trials of 10 min separated by 2 intertrial intervals of 5 min, during which the animals were returned to their home cage. On the first trial, or familiarization phase, the mice were presented with two copies of an unfamiliar object. On the second trial, or place phase, one of the two copies was displaced to a novel location in the arena. On the third trial, or object phase, the copy that had not been moved on the previous trial was replaced by a novel object. Stimuli objects used in all previous experiments were Lego bricks, plastic rings, dice or marbles (size 1.5–3×2–3 cm). The identity of the objects, as well as the spatial location in which these objects were positioned, was balanced between subjects. The number of visits and the time spent exploring each object were scored manually on video recordings. A visit was acknowledged when the nose of the mouse came in direct contact with an object. A percentage of discrimination was calculated for the number of visits and time exploring the objects as following: exploration of displaced or novel objects / total exploration. The discrimination ratio during the familiarization phase was arbitrarily calculated for the object located in the right upper corner of the arena.

Open field. Locomotor activity was monitored at 15 lux either in open fields placed over a white Plexiglas infrared-lit platform. Locomotor activity was recorded via an automated tracking system (Ethovision, Noldus, Wageningen, Netherlands).

Rotarod. The apparatus (Ugo Basile, Germonio, Italy) was set to accelerate from 4 to 40 rpm in 5 min. On day 1, mice were habituated to rotation on the rod at 4 rpm, until they were able to stay more than 180 s. On day 2, mice were tested for three daily trials (60 s intertrial). Each trial started by placing the mouse on the rod and beginning rotation at a constant 4 rpm-speed for 60 s. Then the accelerating program was launched, and the trial ended for a particular mouse when falling off the rod. Time stayed on the rod was automatically recorded.

Blood collection and corticosterone measurements. Mice were euthanized by rapid decapitation 24 h after treatment with Nb20 or vehicle/PBS (48 h after last stressor). Trunk blood was collected in Eppendorf tubes and allowed to clot for 30 min. Serum was isolated after 2000g centrifugation and stored at −20°C. Corticosterone levels were measured using a commercially available ELIZA kit (Thermo Fisher # EIACORT) according to the manufacturer’s instructions.

Immunogenicity analysis. Amino acid sequence of nanobodies was analyzed using NetMHCIIpan version 4.0 to predict binding affinities to MHC class II molecules69. The sequence was submitted in FASTA format to the online tool, and binding predictions were generated for a comprehensive panel of MHC class II alleles. Binding strength was categorized based on percentile ranks, with published thresholds identifying strong, weak, and non-binders.

Data analysis and statistics. The functional data shown represent the average ± SEM of at least 3 individual experiments each performed in triplicate and expressed as means ± standard error means (s.e.m). For the GAP assay, cells with the same transfection conditions were compared in their treatment and a two-way ANOVA test was performed. The non-linear regression curve was used to fit the dose-response curve, giving the IC50 value. For the flow cytometry binding assays, cells transfected with an empty vector were used as a control, and the non-linear regression curve was used to fit the dose response curve, giving the EC50 value. For the SPR, calculation of association (kon) and dissociation (koff) rate constants was based on a 1:1 Langmuir binding model. For the mouse experiments, Marble Burying, Splash Test, Tail Suspension, Elevated Plus Maze and Force Swim Test, comparisons were by one-way ANOVA with Dunnett or Kruskal-Wallis post hoc tests. Assumptions of normality and homogeneity of variance were studied using Shapiro–Wilk tests and Levene’s tests, respectively. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Mean values with s.e.m. are shown. For the ELIZA, a one-way ANOVA test was performed to compare the control treatment with the treated condition. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Mean values with s.e.m. are shown. For the electrophysiology experiments, changes was assessed in a non parametric t-test; the Wilcoxon test. Values of *P<0.05, **P<0.01 and ***P<0.001 ***P<0.0001 were considered to be statistically significant. Calculations, graphs and statistics were generated using GraphPad Prism 9 software (San Diego, CA, USA).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Laboute et al developed a nanobody (Nb20) targeting mGlyR and found that Nb20 functions as an antagonist\nfor mGlyR. Authors performed cell-based analysis, structure determination and animal model experiments to claim that Nb20\nbinds the Cache domain of mGlyR and down-regulates its GAP activity for the anti-depressant effects. The overall idea and\napproaches are sounding and authors showed interesting data in cell-based and animal model analyses. The main\nproblems in this work are in structure analysis part, which are described below. Authors should carefully re-analyze their\nstructures to provide convincing links between the structures and their biological data to improve the quality of this\nmanuscript.\n1. P6, l11 “….we found that the introduction of RGS7/Gb5 accelerated deactivation of its substrate, Gao (Fig.2B,D).\nApplication of Nb20 had no significant effect on either baseline Gao deactivation or RGS7/Gb5-assisted process (Fig.2B,D)”.\nFig 2D, it is not clear where the middle RGS7/Gβ5 (GPR158 –, RGS7/Gβ5 +) data comes from. Unlike those in the left (-/-)\nand right (+/+) in the panel, the source in the middle is unclear. If it is from 2B, the bar graph cannot be same or similar to\nthose of -/-.\n2. Page 6, l -3 “The lack of interplay between Nb20 and glycine likely indicates distinct mechanisms of mGlyR activity\nmodulation by the two ligands and inability of Nb20 to access glycine binding pocket (Supplementary Fig. 4).”\nIn Fig. 4B, Nb20 and glycine do not exhibit a synergistic effect, and there is no available structure of glycine-bound GPR158.\nTherefore, the proposed distinct mechanisms by which Nb20 and glycine require additional supporting evidence.\nFurthermore, on p14: l 5, “Our functional experiments reveal no interaction between glycine and Nb20 effects. While this\nsuggests that Nb20 and glycine may share a similar mechanism of mGlyR inhibition, there is currently no structure of\nglycine-bound mGlyR, and future experiments will need to explore whether Nb20 taps into the mechanism of mGlyR\nantagonism utilized by its endogenous ligand glycine.”\nThis statement is inconsistent with the earlier claim of distinct mechanisms.\n3. P8, “We validated the binding mode of Nb20 by mutating the CDR1 and CDR2 regions involved in the binding which\nresulted in a complete loss of interaction of mutant Nb20 (Nb20*) with mGlyR (Supplementary Fig. 6 & 8).”\nAuthors should provide more detailed information including the mutated residue information to help readers better\nunderstand the texts. Additionally, please explain how Supplementary Figure 6 supports the authors claims.\n4. P8. Structure superposition (Supp Fig 9D and E). Authors should compare with other available structures and perform\nmore careful analysis. To better demonstrate the independent effect of Nb20 on the GAP activity of the mGlyR-RGS7-Gβ5\ncomplex, it would be more appropriate to compare the TM structures between mGlyR-RGS7/Gβ5 and Nb20-mGlyR-\nRGS7/Gβ5.\n5. The structures are not properly analyzed or described in the text. There are many inconsistencies in the authors’\nstructures and their description in the text. These include residue name, number and interactions, and cannot be considered\nas typo. I provided some examples here, but authors should carefully check other parts too.\n(i) P7, l -2. “ .. π-π stacking..between W57 and W162; I am not convinced if they form π-π stacking – please see their\norientation and distance.\n(ii) P8, l 1. There is no N142; S142 and N143 – in any case, both residues are over 7 A away from Y60.\n(iii) P8, l 3, No D198, E198 is far apart from K79 to form a hydrogen bond.\nAlso, considering these features, it is not clear how mutations in these residues affect the interaction between Nb20 and\nmGlyR (Supp Fig 6 and 8).\n6. P9, l2, “…In the mGlyR-RGS7/Gβ5 structure, the ECD domain could not be observed…. the binding of Nb20 stabilized\nboth the ECD and RGS7-Gβ5, enabling the reconstruction of the entire assembly with reasonably well-resolved density.”\nThis is a critical issue as the sentence is not true. The key feature of the authors’model is that Nb20 binds and stabilizes the\nextracellular domain, which was not visible in Nb20-free state. Subsequently the conformational change is relayed to the TM\nand intracellular region, which alters the structure of the RGS7-Gb5. In contrast to authors claim, the extracellular domain,\nTM and RGS7-Gb5 are well defined in the reported Nb20-free mGlyR-RGS7/Gb5 (PDB 7EWP). Because this is the starting\npoint of the authors’model for the structural basis for the Nb20-mediated anti-depressant effect of mGlyR, the\nauthors’proposed model is not consistent with available structures. I strongly suggest that the authors should reinterpret their\ndata by comparing with all available structures including 7EWP.\n7.P9. Even in the structure after focused refinement, the EM density for Nb20 are not well-defined. This is observed from the\npdb and map from the authors and Supplementary Fig. 7D. Thus, one possibility is that the end-regions of the complex\nincluding Nb20 and Gb5 are flexible instead of the Nb20-binding induced release of RGS7-Gb5 from mGlyR. Authors may\nconsider such possibility. Also, the model-fitted-map seems unmatched each other in supplementary Fig. 7D. It would be\nhelpful that authors provide better figure or check their orientations.\n8. P 9. “…Even after local refinement and analysis at various contour levels, the RGS domain density remained\nunresolved…”:\nAuthors may consider to perform structural analysis using a newly generated local map encompassing the 7TM-RGS-Gβ5\nregions. At the current resolution, the map quality is still poor to support claims that the nanobody induced the unresolved\nGβ5 region. Please consider increasing the contour level to reduce noise from detergent micelles. If the signal is\nsufficiently strong, the 7TM-RGS7-Gβ5 complex—excluding the unresolved regions—should remain clearly visible. This would help\ndetermine whether the unresolved areas are due to localized disorder, rather than overall weak density across the entire\nRGS7–Gβ5 region. Then, the authors should represent these maps accordingly. The current maps shown in Supplementary\nFig. 7F appear to be at low contour levels.\n9. P9. Considering the weak density of intracellular region and more than half of the EM density for Gb5 is missing, it is\ndifficult to say 5 A translation of the Gb5 in the presence of Nb20. Description of the conformational change with inaccurate\nstructure could mislead the readers and carefully addressed.\n10. P9. “Nb20 binding disrupts key hydrogen bonds: one between R715 from CTH2 and E172 from RGS7, and another\nbetween T602 from the TM domain and D147 from RGS7 (Supplementary Fig. 9H).”\nT602 and D147 do not form hydrogen bonds in the 7SHF structure. Authors should check this part. Calculate local resolution\nand present it as a figure to claim residue-shift. In my opinion, 4 Å resolution is not sufficient to claim the residue shift.\n11. The FSC curve for the locally refined map (Supplementary Fig. 6G) shows an abnormally large bump, suggesting\noverfitting due to an overly tight mask. One of the binary masks shown in Supplementary Fig. 5 also appears too tight,\nexcluding signal near the edge regions. The authors should consider to repeat the local refinement using a new mask\ngenerated from a much larger base mask, for example using ChimeraX.\n12. Overall resolution is insufficient to claim the loop shifts or residue shifts (Supplementary Fig. 9D, E, H). The authors are\nhighly encouraged to present model-fitted-maps for all regions separately.\nMinor comments\n1. P7 l9, “To improve the map, local refinements using soft mak corresponding to ECD-Nb20”\nTypo, refiments > refinements, soft mak > soft mask.\n2. Supplementary Fig. 9C and Supplementary Fig. 9F. Please indicate the the position of the stalk domain.\n3. Are Fig. 3C and 3D in the same view? It would be helpful to match their viewpoints for easier comparison. Additionally,\nindicating the positions of CDR1 and CDR2 would help readers to interpret the figure.", "reviewer_2": "The revised manuscript has significantly improved and addresses my previous main concerns. The inclusion of additional\nbehavioral tests strengthens the interpretation of Nb20’s effects on depression-related phenotypes. Importantly, the authors\nnow provide data on hedonic behavior, sex differences, and corticosterone levels following administration. Clarifications\nregarding the EPM findings over time and additional behavioral parameters such as arm entries and head-dips have also\nbeen included and are clearly presented. I find the revisions acceptable, and I believe the manuscript is now suitable for\npublication.", "reviewer_3": "The authors develop a new nanobody-based strategy to antagonize the mGlyR, which rescues behavioral deficits\nassociated with chronic stress and psychiatric disease. The authors show that the Nb20 nanobody selectively modulates\nmGlyR function and defines the downstream biochemical and structural mechanisms, as well as the electrophysiological\nconsequences. Multiple delivery approaches of Nb20 induce antidepressive-like and long-lasting phenotypes in a variety of\nbehavioral assays. These behavioral effects are associated with increased excitability of neurons in the prelimbic cortex,\nwhere mGlyR is highly expressed. This work reflects a new and exciting angle to target brain-specific antigens, with\nintranasal delivery of a nanobody highlighting translational potential. I have a few suggestions that would strengthen the\ncentral claims of the manuscript.\n1.) Is the Nb20 antibody specific to mGlyR in vivo? The null nanobody controls for off-target effects of the surgery and\nnanobody domain, but does not rule out promiscuous binding of Nb20 to other targets, which could generate indirect effects\non behavior. Since the authors studied mGlyR knockout mice, are the antidepressant-like behavioral effects of Nb20 affected\nin mice lacking the expected antigen?\n2.) In Fig 5, the authors confirm that Nb20-based mGlyR antagonism phenocopies the increased excitability of L2/3 PFC\nneurons in mGlyR KO mice, which is a valuable control. However, it’s not clear how this relates to the behavioral effects\nobserved after Nb20 treatment in Fig 4 since the treatment timescales differ significantly (10 min treatment in vitro in Fig 5 vs\n24+ hrs in vivo in Fig 4) and these effects may be correlative. If the authors performed these electrophysiology experiments\nfrom mice treated with Nb20 (as in Fig 4), are these neurons more excitable? Alternatively, are proxies of excitability, such\nas cFos, increased in mice treated with Nb20?\n3.) While the results are indeed exciting, the manuscript appears overly hyperbolic in several areas. For example, in the\ndiscussion they “report a unique immunotherapy solution for a major neuropsychiatric condition- major depressive disorder”,\nbut depression reflects a human condition with environmental drivers that are not addressed in this manuscript. Additionally,\nthe authors claim immunotherapies offer “low toxicity”, but one of the current limitations of immunotherapies is toxicity,\npartially due to an incomplete mechanistic understanding (which is highlighted by points 1 and 2 above)."}, "author_response": "We appreciate continuing interest of the reviewers in this work and many helpful suggestions.\nWe have incorporated all of feedback from the reviewers in the revised manuscript and are\ngrateful for reviewer’s time and help in improving this manuscript. Our responses to specific\nreviewer’s comments are as follows. Changes in the manuscript are highlighted in blue font.\nReviewer #1\nComment 1. P6, l11 “….we found that the introduction of RGS7/Gb5 accelerated\ndeactivation of its substrate, Gao (Fig.2B,D). Application of Nb20 had no significant effect\non either baseline Gao deactivation or RGS7/Gb5-assisted process (Fig.2B,D)”.\nFig 2D, it is not clear where the middle RGS7/Gβ5 (GPR158 –, RGS7/Gβ5 +) data comes\nfrom. Unlike those in the left (-/-) and right (+/+) in the panel, the source in the middle is\nunclear. If it is from 2B, the bar graph cannot be same or similar to those of -/-.\nResponse: We apologize for not making presentation of these data consistent. In fact, we\ncollected the entire dataset with all conditions (baseline, No RGS, No mGlyR) with or\nwithout Nb20 in parallel. However, the Figure as presented showed a subset of this data as\nrepresentative traces in panels B an C and another subset as quantification in panel D. We\nagree that this is confusing. To rectify it, we are now showing the entire dataset aligning all\nthe conditions between panels B, C and D. Thank you for bringing it to our attention.\nComment 2. Page 6, l -3 “The lack of interplay between Nb20 and glycine likely indicates\ndistinct mechanisms of mGlyR activity modulation by the two ligands and inability of Nb20\nto access glycine binding pocket (Supplementary Fig. 4).”\nIn Fig. 4B, Nb20 and glycine do not exhibit a synergistic effect, and there is no available\nstructure of glycine-bound GPR158. Therefore, the proposed distinct mechanisms by which\nNb20 and glycine require additional supporting evidence.\nFurthermore, on p14: l 5, “Our functional experiments reveal no interaction between glycine\nand Nb20 effects. While this suggests that Nb20 and glycine may share a similar mechanism\nof mGlyR inhibition, there is currently no structure of glycine-bound mGlyR, and future\nexperiments will need to explore whether Nb20 taps into the mechanism of mGlyR\nantagonism utilized by its endogenous ligand glycine.”\nThis statement is inconsistent with the earlier claim of distinct mechanisms.\nResponse: We agree with the reviewer that our treatment of Nb20 vs glycine effects have\nbeen confusing. The difficulty is largely explained by the lack of the glycine bound structure\nof mGlyR. We feel that in the absence of such structure any claims regarding the similarity or\ndifferences between Nb20 and glycine in terms of the mechanism of action will be highly\nspeculative. With this, we decided to remove the preliminary data on the combinatorial\neffects of Nb20 and glycine reserving thorough investigation on the mechanisms to the future\nwhen glycine bound structure of mGlyR becomes available and/or we interrogate their\ninteraction with focused biophysical and biochemical approaches which seem to fall outside\nof the focus of this study.\nComment 3. P8, “We validated the binding mode of Nb20 by mutating the CDR1 and CDR2\nregions involved in the binding which resulted in a complete loss of interaction of mutant\nNb20 (Nb20*) with mGlyR (Supplementary Fig. 6 & 8).”\nAuthors should provide more detailed information including the mutated residue information\nto help readers better understand the texts. Additionally, please explain how Supplementary\nFigure 6 supports the authors claims.\nResponse: The information regarding the exact residues mutated to generate control NB20*\nhas been provided in the Methods section. We have now revised the main text to provide\nbetter description and reference the Methods section. We apologize for inadvertently calling\nout Supplementary Fig 8 in this section – it is indeed irrelevant to the point regarding Nb20*\ndesign or performance.\n4. P8. Structure superposition (Supp Fig 9D and E). Authors should compare with other\navailable structures and perform more careful analysis. To better demonstrate the independent\neffect of Nb20 on the GAP activity of the mGlyR-RGS7-Gβ5 complex, it would be more\nappropriate to compare the TM structures between mGlyR-RGS7/Gβ5 and Nb20-mGlyR-\nRGS7/Gβ5.\nResponse: Thank you for this valuable suggestion. We have now performed a transmembrane\n(TM) region–based structural comparison between the mGlyR–RGS7/Gβ5 complex and the\nNb20-bound mGlyR–RGS7/Gβ5 complex, as well as with other available related structures\n(PDB:7EWP). The revised text and Supplementary Fig. 9F, H (Now Supplementary Figure\n8) have been updated accordingly (page 8).\n5. The structures are not properly analyzed or described in the text. There are many\ninconsistencies in the authors’ structures and their description in the text. These include\nresidue name, number and interactions, and cannot be considered as typo. I provided some\nexamples here, but authors should carefully check other parts too.\n(i) P7, l -2. “ .. π-π stacking..between W57 and W162; I am not convinced if they form π-π\nstacking – please see their orientation and distance\nResponse: Thank you for pointing this out. Upon re-examining the structural data, we agree\nthat the side chain orientations and inter-residue distance are not fully consistent with a\ncanonical π–π stacking interaction. We have revised the statement accordingly to clarify that\nW57 and W162 are in close spatial proximity with a potential for interactions rather than\nspecifying a defined π–π stacking interaction (page 7).\n(ii) P8, l 1. There is no N142; S142 and N143 – in any case, both residues are over 7 A away\nfrom Y60.\nResponse: Thank you for pointing this out. We apologize for the oversight regarding the\nresidue numbering. Upon re-examination, we confirm that it is N143 (not N142) whose side\nchain forms a hydrogen bond with the backbone carbonyl of T58 in Nb20. We have corrected\nthe residue numbering and revised the text accordingly.\n(iii) P8, l 3, No D198, E198 is far apart from K79 to form a hydrogen bond.\nAlso, considering these features, it is not clear how mutations in these residues affect the\ninteraction between Nb20 and mGlyR (Supp Fig 6 and 8).\nResponse: Thank you for pointing this out. Upon re-examination, we confirm that it is E198\n(not D198) whose side chain forms a hydrogen bond with N31 of Nb20. We have corrected\nthe residue numbering and revised the text accordingly. Furthermore, we have clarified in the\nmanuscript how mutations at these positions potentially alter the local interaction network\n(page 8) and thereby influence Nb20 binding to mGlyR, as illustrated in Supplementary Fig\n7.\nComment 6. P9, l2, “…In the mGlyR-RGS7/Gβ5 structure, the ECD domain could not be\nobserved…. the binding of Nb20 stabilized both the ECD and RGS7-Gβ5, enabling the\nreconstruction of the entire assembly with reasonably well-resolved density.”\nThis is a critical issue as the sentence is not true. The key feature of the authors’ model is that\nNb20 binds and stabilizes the extracellular domain, which was not visible in Nb20-free state.\nSubsequently the conformational change is relayed to the TM and intracellular region, which\nalters the structure of the RGS7-Gb5. In contrast to authors claim, the extracellular domain,\nTM and RGS7-Gb5 are well defined in the reported Nb20-free mGlyR-RGS7/Gb5 (PDB\n7EWP). Because this is the starting point of the authors’model for the structural basis for the\nNb20-mediated anti-depressant effect of mGlyR, the authors’proposed model is not\nconsistent with available structures. I strongly suggest that the authors should reinterpret their\ndata by comparing with all available structures including 7EWP.\nResponse: We thank the reviewer for this important comment. At higher Cryo-EM\nresolutions, regions of intrinsic flexibility tend to appear more disordered or diffuse in the\ndensity map. This is because the electron density reflects the ensemble of conformations\nsampled by these flexible regions rather than a single static state. While high-resolution maps\nprovide detailed features (such as side chains) for well-ordered, rigid regions, they also make\nthe conformational heterogeneity of flexible domains more evident. Instead of a well-defined\ndensity, flexible domains display an averaged-out signal across multiple conformations,\nresulting in a blurred or “scattered” appearance.\nThis effect is clearly illustrated in our study through 3D variability analysis, which shows that\nthe extracellular domain (ECD) undergoes significant motion and is highly flexible. Such\nheterogeneity explains why the ECD appears poorly resolved despite the overall map\nresolution being high. A similar phenomenon is seen in the previously solved mGlyR\nstructure 7EWP (EMDB 31360) at 4.3 Å resolution: the transmembrane (TM) region shows\nclear side-chain features, whereas the ECD density lacks side-chain information, indicating\nintrinsic flexibility in this region even at lower resolution. When structures are refined to\nhigher resolutions—as in our case (3.3 Å)—the TM region becomes even better defined, but\nthe flexible ECD appears even more scattered due to high-resolution filtering revealing its\nconformational heterogeneity.\nTo address the reviewer’s suggestion, we have expanded our structural comparison to include\nPDB 7EWP and other available mGlyR structures. This updated analysis is incorporated in\nthe revised Results (page 8, Supplementary Fig. 8F, H), where we describe the\nconformational differences observed.\nComment 7. P9. Even in the structure after focused refinement, the EM density for Nb20 are\nnot well-defined. This is observed from the pdb and map from the authors and Supplementary\nFig. 7D. Thus, one possibility is that the end-regions of the complex including Nb20 and Gb5\nare flexible instead of the Nb20-binding induced release of RGS7-Gb5 from mGlyR. Authors\nmay consider such possibility. Also, the model-fitted-map seems unmatched each other in\nsupplementary Fig. 7D. It would be helpful that authors provide better figure or check their\norientations.\nResponse: We thank the reviewer for this insightful observation. We agree that, even after\nfocused refinement, the EM density for Nb20 is not well defined in our map, consistent with\nthe reviewer’s assessment of our PDB, EM map. As suggested, one plausible explanation is\nthat the peripheral components of the complex, including Nb20 and Gβ5, exhibit higher\nflexibility, which contributes to the diffuse density observed. We have now included this\npossibility in the revised Discussion section (pages 9), noting that the observed density does\nnot necessarily imply complete release of the RGS7–Gβ5 module from mGlyR upon Nb20\nbinding. Regarding the reviewer’s comment about Supplementary Fig. 7D (Now\nSupplementary Fig. 6D), we have carefully re-examined the map-to-model fit and the\norientations presented. The figure has been updated with improved orientation and rendering\nand clearer depiction of the model fitting, ensuring that the map and model are accurately\naligned and more easily interpretable. The revised figure is provided as Supplementary Fig.\n6D in the updated manuscript.\nComment 8. P 9. “…Even after local refinement and analysis at various contour levels, the\nRGS domain density remained unresolved…”:\nAuthors may consider to perform structural analysis using a newly generated local map\nencompassing the 7TM-RGS-Gβ5 regions. At the current resolution, the map quality is still\npoor to support claims that the nanobody induced the unresolved Gβ5 region. Please consider\nincreasing the contour level to reduce noise from detergent micelles. If the signal is\nsufficiently strong, the 7TM-RGS7-Gβ5 complex—excluding the unresolved regions—\nshould remain clearly visible. This would help determine whether the unresolved areas are\ndue to localized disorder, rather than overall weak density across the entire RGS7–Gβ5\nregion. Then, the authors should represent these maps accordingly. The current maps shown in\nSupplementary Fig. 7F appear to be at low contour levels.\nResponse: We thank the reviewer for these constructive suggestions. We have performed\nadditional local refinements focusing on the 7TM–RGS7–Gβ5 regions and generated new\nlocally refined maps to better evaluate the density in these areas. We also examined the maps\nat various contour levels, including higher thresholds to minimize noise contributions from\ndetergent micelles. As suggested, we now provide updated figures (revised Supplementary\nFig. 6F) where the density corresponding to the well-resolved regions of the 7TM–RGS7–\nGβ5 complex remains visible even after increasing the contour level. These analyses confirm\nthat the lack of resolved density for portions of the RGS7–Gβ5 region is likely due to\nlocalized conformational flexibility rather than an overall weak signal across the entire\nmodule. The revised results sections (page 9) now clarify this interpretation, and the updated\nmaps are represented accordingly.\nComment 9. P9. Considering the weak density of intracellular region and more than half of\nthe EM density for Gb5 is missing, it is difficult to say 5 A translation of the Gb5 in the\npresence of Nb20. Description of the conformational change with inaccurate structure could\nmislead the readers and carefully addressed.\nResponse: We thank the reviewer for pointing this out. We agree that the intracellular region\nexhibits weak density and that the EM density for Gβ5 is only partially resolved, which\nmakes it challenging to directly interpret small positional shifts with high confidence. In light\nof this, we have revised the text to present the 5 Å translation of Gβ5 in the presence of Nb20\nmore cautiously. Rather than describing it as a definitive conformational change, we now\ndiscuss it as a plausible movement inferred from the available density, while clearly\nacknowledging the limitations arising from the incomplete map and flexibility of the\nintracellular module.\nComment 10. P9. “Nb20 binding disrupts key hydrogen bonds: one between R715 from\nCTH2 and E172 from RGS7, and another between T602 from the TM domain and D147 from\nRGS7 (Supplementary Fig. 9H).”\nT602 and D147 do not form hydrogen bonds in the 7SHF structure. Authors should check this\npart. Calculate local resolution and present it as a figure to claim residue-shift. In my opinion,\n4 Å resolution is not sufficient to claim the residue shift.\nResponse: We thank the reviewer for pointing this out. Upon re-examination of the 7SHF\nstructure and our refined maps, we agree that T602 and D147 do not form a direct hydrogen\nbond. We have corrected this statement in the manuscript. Instead, we now describe the\ninteractions more cautiously without overinterpreting the density at this resolution. At the\ncurrent ~4 Å resolution, we agree that the evidence is insufficient to support a definitive\nresidue shift; therefore, we have revised the discussion to reflect this limitation (Page 9).\nComment 11. The FSC curve for the locally refined map (Supplementary Fig. 6G) shows an\nabnormally large bump, suggesting overfitting due to an overly tight mask. One of the binary\nmasks shown in Supplementary Fig. 5 also appears too tight, excluding signal near the edge\nregions. The authors should consider to repeat the local refinement using a new mask\ngenerated from a much larger base mask, for example using ChimeraX.\nResponse: We thank the reviewer for this helpful comment. We agree that the binary mask\nused in the initial refinement was too tight, which likely contributed to the bump observed in\nthe FSC curve of the locally refined map (Supplementary Fig. 5G). Following the reviewer’s\nsuggestion, we generated a more generous mask and repeated the local refinement. This\nadjustment significantly reduced the bump in the FSC curve and yielded more reliable\nrefinement statistics. The revised masks and updated FSC curves are now provided in\nSupplementary Fig. 5G.\nComment 12. Overall resolution is insufficient to claim the loop shifts or residue shifts\n(Supplementary Fig. 9D, E, H). The authors are highly encouraged to present model-fitted-\nmaps for all regions separately.\nResponse: We agree with the reviewer that the local resolution is limited in some regions, and\ntherefore caution is required when interpreting residue- or loop-level shifts. In the revised\nmanuscript, we have toned down our claims regarding side chain or residue movements and\ninstead describe the changes at the level of secondary structure or domain rearrangements. In\naddition, as suggested, we now provide model-fitted maps for the corresponding regions\n(Supplementary Fig. 6A, B, C), which allow readers to better assess the local map quality and\nthe basis for our structural interpretation.\nMinor comments\n1. P7 l9, “To improve the map, local refinements using soft mak corresponding to ECD-\nNb20”\nTypo, refiments > refinements, soft mak > soft mask.\nResponse: We apologize for the typographical errors and we have corrected them\n2. Supplementary Fig. 9C and Supplementary Fig. 9F. Please indicate the position of the stalk\ndomain.\nResponse: We have now indicated the position of the stalk domain in Supplementary Fig. 8C\nand Supplementary Fig. 8F for clarity.\n3. Are Fig. 3C and 3D in the same view? It would be helpful to match their viewpoints for\neasier comparison. Additionally, indicating the positions of CDR1 and CDR2 would help\nreaders to interpret the figure.\nResponse: We have adjusted the views to better illustrate the interactions and have now\naligned them as closely as possible to match the perspective shown in Fig. 3C, while still\nmaintaining the key interactions.\nReviewer #1\nComment 1.) Is the Nb20 antibody specific to mGlyR in vivo? The null nanobody controls\nfor off-target effects of the surgery and nanobody domain, but does not rule out promiscuous\nbinding of Nb20 to other targets, which could generate indirect effects on behavior. Since the\nauthors studied mGlyR knockout mice, are the antidepressant-like behavioral effects of Nb20\naffected in mice lacking the expected antigen?\nResponse: We think that we established specificity of the Nb20 effects beyond reasonable\ndoubt. The behavioral effects that we observe in response to Nb20 administration match very\nwell the phenotypes seen in mGlyR KO mice. In brain slices, Nb20 produces the same effect\non excitability seen upon genetic elimination of mGlyR. Finally, we use a very stringent\ncontrol – the same Nb mutated not to interact with its target mGlyR and see no effects with it.\nOn the sum, this is beyond what is typically done to establish the specificity of a biologic.\nUnlike small molecules, nanobodies and antibodies are usually very target selective and\nsignificant engagement of other targets at low concentration we use amidst extremely high\naffinity for the intended target are not commonly expected. The experiment proposed by the\nreviewer to evaluate the effect of Nb20 in mGlyR KO mice is great in spirit but unlikely to be\ninformative. mGlyR KO mice show strong baseline anti-depressant phenotype- the same\neffect produced by the Nb20 treatment – thus their anti-depressant responses can’t be\nevaluated due to “floor/ceiling” effect. However, we understand that one can never be certain\nabout lack of off-target activities. Thus, we revised the manuscript to indicate this caveat in\nthe Discussion section (page 14).\nComment 2.) In Fig 5, the authors confirm that Nb20-based mGlyR antagonism phenocopies\nthe increased excitability of L2/3 PFC neurons in mGlyR KO mice, which is a valuable\ncontrol. However, it’s not clear how this relates to the behavioral effects observed after Nb20\ntreatment in Fig 4 since the treatment timescales differ significantly (10 min treatment in\nvitro in Fig 5 vs 24+ hrs in vivo in Fig 4) and these effects may be correlative. If the authors\nperformed these electrophysiology experiments from mice treated with Nb20 (as in Fig 4),\nare these neurons more excitable? Alternatively, are proxies of excitability, such as cFos,\nincreased in mice treated with Nb20?\nResponse: Electrophysiology experiments were designed to test whether inhibition of mGlyR\nwith Nb20 would produce similar effect as mGlyR knockout or its inhibition by glycine\nestablishing the effects at a more mechanistic level. We understand the reviewer’s point and\ncertainly agree that recording electrophysiological properties in neurons of mice following\nNb20 administration and behavioral evaluation would have been ideal and very powerful.\nHowever, these experiments are extremely challenging and difficult to interpret due to several\nreasons. The main problem and the biggest limitation are the complexity of the stress\nresponses in terms of their effects on the neuronal activity of cell populations and circuits\nwhich we found to be highly variable. This is not endemic to our study and in fact the effects\nof stress on circuits and neuronal activity is a hotly debated area with a range of disparate\neffects described (e.g. PMIDs: 18923511, 28856337, 34893785). Thus, what may appear as a\nsimple follow up in a context of a Nb20 treatment in fact requires defining stable effects of\nstress on neural circuits which is a momentous feat even if one looks at “simpler” proxies\nsuch as cFos staining. We appreciate reviewer’s understanding that such experiments might\nnot be feasible for us to accomplish this in a framework of a study that mostly geared towards\natomic/molecular angle of understanding how Nb20 alter mGlyR. We further acknowledged\nthese limitations and the need to better define physiological effects of Nb20 on neural circuits\nand neuronal properties in the future studies.\n3.) While the results are indeed exciting, the manuscript appears overly hyperbolic in several\nareas. For example, in the discussion they “report a unique immunotherapy solution for a\nmajor neuropsychiatric condition- major depressive disorder”, but depression reflects a\nhuman condition with environmental drivers that are not addressed in this manuscript.\nAdditionally, the authors claim immunotherapies offer “low toxicity”, but one of the current\nlimitations of immunotherapies is toxicity, partially due to an incomplete mechanistic\nunderstanding (which is highlighted by points 1 and 2 above).\nResponse: We certainly understand the limitations and arduous task of translating the\nobservations in rodents to humans. We have tried acknowledging these limitations in the\nmanuscript and have done more of this during this revision (e.g. page 13). We also revised the\nmanuscript to subdue hyperbolic claims.\nWe appreciate reviewer’s help in improving our manuscript. We have revised the manuscript to\naddress outstanding issues. Our responses to specific concerns are as follows.\n1.. Supple Fig 6 a, c, d, f are still messy and too complicated.\n(i) Authors should provide better views or sections of the maps and models. Fig S6f. Even at very low\ncontour level (near background) of the map where micelle completely covers the TMs, very poor\ndensity for RGS7 is shown. I do not think it is proper to build the model here. Accordingly,\ndescription of the conformation changes in RGS7 is unlikely to be accurate (as density for the\nsecondary structures for RGS7 is unclear) and should be removed.\nResponse: Based on the suggestions of the reviewer we have improved the view for the TM domain\nand the RGS7 domains. As demonstrated in our previous revision using 3D variability analysis, both\nthe RGS7 and ECD domains exhibit substantial mobility relative to the transmembrane domain.\nConsequently, these regions remain at lower local resolution, although their overall positions could\nstill be defined through rigid-body docking and refinement. Moreover, we do observe clear, albeit\nlow-feature, density corresponding to RGS7 and only the global shape profile Gβ5 (hence it was rigid\nbody docked into the map). We included them in the model through rigid-body fitting to provide a\ncomplete and accurate representation of the complex architecture. The DEP/DEX domain of RGS7,\nwhich directly contacts the receptor, is shown in better view, showing the secondary structure quite\nvisible in Supplementary Figure 6. The RGS domain remained invisible in the map and hence was not\ndocked into the maps. We do see garbled density corresponding to Gβ5 and hence, it was docked into\nthe map and was not built further due to lack of sufficient information.\n(ii) Also, in FigS6f, some TMs do not form helices but rather they form loops (see figure S6f).\nResponse: This issue arose due to an incorrect interpretation of the PDB file by Chimera, which\ndisplayed certain transmembrane helices as loops. We have now corrected this visualization error, and\nthe updated Supplementary Fig. S6f properly shows these regions as continuous α-helices within the\nwell-defined transmembrane domain.\n(iii) Method section: Authors described “…manual building..”. How RGS7 – Gb5 can be manually\nbuilt? Authors should more precisely describe what they have done. Also, authors should describe\nwhich parts of the models are visible in the maps either in the main text or methods section. In\nprinciple, model should not fit in the invisible region.\nResponse: The Methods section has been revised to more precisely describe the model-building\nprocedure. We now specify that only the well-defined regions of the transmembrane domain were\nmanually adjusted in Coot based on visible cryo-EM density, while Gβ5 and RGS7 were docked into\nthe map as rigid bodies. The RGS domain, which is not visible in the map, was not built and remains\nabsent from the final model. We cannot leave the density observed at very low contour levels entirely\nunaccounted for; therefore, we docked RGS7 and Gβ5 into this region based on our prior structural\ninformation, which confirms that the corresponding density arises from these components. The\nrevised Methods section explicitly describes that the transmembrane region was built de novo into the\ndensity, whereas RGS7 and Gβ5 were docked and refined as rigid bodies. Additionally, we have\nclarified that no part of the model was fitted into regions lacking interpretable density.\n2. P7, line 2 and supple Fig 4 & 5,\n(i) the resolution is unlikely to be 3.83 A. In S5D, the GSFSC curve decreases and increases again\nnear 0.143 (unusual type of curve), and this is unlikely to be the true resolution.\nResponse: We appreciate the reviewer’s careful examination of the GSFSC curve. We also noticed the\nunusual dip in the curve near the 0.143 threshold during multiple steps of data processing. We believe\nthis feature is intrinsic to the dataset and may arise from the non-uniform distribution of particle\norientations or conformational flexibility within the complex, the exact reason for this dip is far from\nunderstood. Despite this minor anomaly, the overall map quality supports a global resolution of 3.89\nÅ. The transmembrane domain, in particular, exhibits well-defined density with clearly visible side\nchains, consistent with this resolution. We note, however, that the RGS and ECD domains are\nresolved at lower local resolution due to their inherent flexibility, as also indicated in variability\nanalysis.\n(ii) Authors wrote 3.83 A in the text, but 3.89 A in the figure section – which one is correct? Also 3.43\nvs 3.49 ? in S5D.\nResponse: We thank the reviewer for noticing this inconsistency. The discrepancies in the reported\nresolutions have been corrected throughout the text and figures for consistency.\n(iii) Also, authors should provide the local resolution maps for the structure with different colors.\nPossibly, they may represent maps including local resolution at the bottom of Fig S4.\nResponse: The figure has been updated to include local resolution maps rendered in color, illustrating\nthe resolution variation across the structure. The local resolution representation has also been added at\nthe bottom of Supplementary Fig. S4 as requested.\n3. The focused refined EM density for the Nb20-ECD is not sufficient to fit the side chains in most of\nthe regions. Therefore, description of the interface interaction could provide wrong information. Some\nexamples include;\n(i) E198 – N31; shorter one has 4.5 A distance, the other one is over 9 A and both are too far.\nMoreover, carbonyl - carboxyl oxygen in shorter one\nResponse: We agree that the focused-refined map for the Nb20–ECD complex does not allow\nunambiguous placement of all side chains at high confidence because this interface can not be\nresolved with higher detail in our structures presented. We have therefore moderated our description\nof the Nb20–ECD interface in the text to avoid overinterpretation and caution. The interaction of\nE198–N31 have been re-evaluated, and all specific atomic interactions that were not reliably\nsupported by the map have been removed from the manuscript.\n(ii) between R56 of Nb20 and E166 of subunit A: density for the R56 side-chain is not visible unless\nyou lower the contour level near the background level.\nResponse: We acknowledge that the side-chain density for R56 of Nb20 is weak and becomes visible\nonly at lower contour levels. Accordingly, we now note in the text and figure legend that the side-\nchain positioning in this region is tentative and should be interpreted with caution.\n(iii) Nb20* mutant: the mutation study performed here is more likely complete disruption of the local\ncore structure CDR1 and CDR2 rather than disruption of the interaction by the surface mutation. I do\nappreciate if authors have attempted to provide clearer mutational effects, but at least brief comment\nwould help.\nResponse: Regarding the Nb20* mutant, we appreciate the reviewer comment. We agree that the\nmutations introduced may affect the local core structure of CDR1 and CDR2, potentially influencing\nNb20 more globally in addition to the interface interaction. We have added a brief statement\ndiscussing this possibility in the revised Results section.\nAlso, in method section: Nb20*: residue number “29 to 35” vs “30 – 35” in the maintext & others.\nThese numbers are inconsistent number with PDB, which are the correct numbers?\nWe thank the reviewer for pointing out the inconsistency in residue numbering. The numbering has\nbeen carefully cross-checked with the PDB file, and all references are now consistent across the main\ntext, Methods section, and figure legends. The correct residue range for Nb20* is residues 30–35 and\nhas been changed accordingly in the text and figures.\n4. P 8 “.. mGlyR– RGS7–Gβ5 (PDB: 7SHF, 7EWP) with that of the Nb20-bound complex. While the\noverall TM domains are largely conserved, notable deviations of up to 5.5 A were observed in TM3,\nTM4, and the connecting loop (Supplementary Fig. 8F, H)…”\nI have compared the TMs (TM3, TM4 and other TMs) and linkers between the current model\n(mGlyR-Nb20-RGS-Focused2.pdb) and 7EWP by aligning entire TMDs, and found out no notable\ndifferences. In fact, the largest differences in TM3 , TM4 and connecting loops are near or less than 1\nA. Authors made inaccurate description and must correct or delete the sentence.\nResponse: We thank the reviewer for the analysis and for pointing out the difference observed when\naligning the entire TMD. As originally suggested by the reviewer, we have included PDB: 7EWP in\nour comparison of the transmembrane domains. Upon re-analysis, we confirm that the deviation\nbetween the mGlyR–Nb20–RGS–Focused2 model and 7EWP is indeed small (approximately 1.2 Å),\nconsistent with the reviewer’s observation.\nHowever, the reviewer has to also consider the comparison with our previously published structure\n(PDB: 7SHF). In this comparison, we observe a larger local deviation of up to 5.5 Å, particularly near\nresidue T514 in TM3. This difference reflects subtle but conformational variability among the\navailable mGlyR–RGS7–Gβ5 complexes, rather than any modelling inaccuracy (Supplementary Fig.\n8F).” Thus, we disagree with labelling of our interpretation as “inaccurate”.\nTo ensure accuracy and fairness, we have revised the manuscript to clearly state the range of\ndeviations observed across all available structures (7EWP, 7SHF, and the current model) and to clarify\nthat the higher deviation originates from the comparison with 7SHF.\n5. “that this domain becomes highly flexible upon binding the Nb20 to ECD. Superimposition of\nNb20-bound and free structures by the 7TM region suggested plausible changes……that remodeling\nat the 7TM interface triggers changes in contacting residues of…””\nSince there are no noticeable changes in TM, this sentence is not correct. Considering TMD retains\nsame structure upon Nb20 binding, authors should reconsider their proposed model.\nResponse: We thank the reviewer for the comment. In light of our previously published structure\n(PDB ID: 7SHF), in addition to comparison with 7EWP, we maintain that the observed flexibility of\nthe domain upon Nb20 binding is supported. However, we agree that the original phrasing could be\nmisleading regarding the transmembrane domain. We have therefore rephrased the sentence to clarify\nthat while the 7TM region remains largely unchanged, binding of Nb20 to the ECD induces local\nflexibility in the extracellular and RGS domains, which may modulate interactions at the interface.\n6. “In the mGlyR-RGS7/Gβ5 structures, the ECD domain was highly flexible, resulting in low\nresolution features as observed in 7EWP or scattered density at relatively higher resolution in 7SHF\nstructures. The binding of Nb20 appeared to stabilize both the ECD and RGS7-Gβ5 module, yielding\nrelatively well-resolved density for ECD. Strikingly, comparison revealed that the density\ncorresponding to the RGS domain, which typically wraps around Gβ5 from below, was poorly\nresolved in the Nb20-bound structure, suggesting that RGS domain adopts”\nThis is not properly revised paragraph. This is only to justify their claims that Nb20 stabilizes ECD.\nResolution of the 7EWP ECD is not sufficient to place the side-chain, but main-chain for ECD is well\ndefined. Nb20-bound EM map is no better (to place the side-chain) than the Nb-free map (7EWP) to\nclaim that Nb20 stabilizes ECD of mGlyR.\nResponse: We agree that the current resolution of both the Nb20-bound and Nb20-free (7EWP) maps\ndoes not allow reliable placement of ECD side chains. However, in light of our previously published\nstructures, where the ECD was not observed upon binding the RGS complex, the presence of density\nfor both the ECD and RGS complex under our experimental conditions hints at a potential stabilizing\neffect of Nb20 on the ECD. This observation differs from the 7EWP structures reported by other\ngroups. Keeping these points in mind, we have revised the paragraph to modify statements suggesting\nthat Nb20 improves ECD stability. The updated text now focuses on the observed map features\nwithout overinterpretation of the effects of Nb20 binding.\n(i) low resolution vs high resolution: Description of resolution is meaningful only if the features are\npresent and can be comparable. This sentence gives impression that the ECD of 7EWP is lower\nresolution than that of 7SHF. However, 7SHF does not have ECD, a key feature authors described.\nComparison of the local resolution of ECDs between 7EWP and 7SHF would be reasonable. Thus,\nthere is no point of describing resolution here, and should be removed as this type of description only\nconfuses the readers.\n(ii) For the readers to clearly understand, the references for 7EWP and 7SHF should be included.\nResponse: We thank the reviewer for the suggestion. References for PDB entries 7EWP and 7SHF\nhave been included in the text to ensure clarity for the readers.\n(iii) After careful checking of the cryo-EM map for the ECD in Nb20-free and Nb20-bound, I cannot\nagree with authors claims that Nb20-binding stabilizes ECD and their further idea.\nResponse: While we agree that the maps do not allow unambiguous placement of ECD side chains\nand the original claim was overstated, we do observe density for the ECD in the RGS7–Gβ5–bound\ncomplex in the presence of Nb20, which was not observed in our previous structures in presence of\nRGS7. This contrasts with our previously published structures, where the ECD was not visible when\nthe RGS complex was bound, suggesting a potentially improving the map corresponding to ECD in\npresence of RGS7-Gbeta5 bound.\nAccordingly, we have moderated our claim in the revised text to avoid overstatement. We now state\nthat Nb20 may contribute to stabilizing the ECD in the presence of RGS7–Gβ5, while focusing on the\nobserved map features rather than over-interpreting the effect of Nb20 binding.\n7. “..but relatively low resolution in the ECD and Nb20 binding regions. Nevertheless,\nthese maps allowed us to construct the complete models of the mGlyR-Nb20 and mGlyR-RGS\ncomplexes,”.\nThe map for the RGS complex is poorly defined and not sufficient to build the complete model. As I\ncommented above, it is not possible to trace the main-chains for RGS7 and Gb5. Thus, authors should\nrevise this part.\nResponse: We thank the reviewer for the comment. We agree that the density for the RGS7–Gβ5\nregion is relatively poorly defined and does not allow manual tracing of main chains. Accordingly, we\nhave revised the text to clarify that the RGS7–Gβ5 module was rigid-body docked and refined into the\ncryo-EM density rather than manually adjusted in Coot. The observed density corresponds to this\nregion, and we cannot leave it unaccounted for, as its assignment to RGS7–Gβ5 is well supported by\nour previous structural knowledge. The revised description now accurately reflects the modelling\napproach used for this flexible region.\n8. Table 1 for the statistics for the structural analysis. Why only two PDBs are reported? Are authors\nnot going to deposit the focused-refined structures? Also, authors should clearly describe which data\nwill be available to public with their statistics.\nResponse: The focused refined structures do not enhance the local resolution significantly for the\nECD or the RGS7-GB5 regions which were poorly resolved in the consensus refined structure\ntherefore we have deposited just two PDBs (Nb20-mGlyR, Nb20-mGlyR-RGS7-Gβ5 ) and the\ncorresponding maps (EMDB-65225, EMDB-65226)\nThank you for evaluating our revised manuscript. We have adjusted the manuscript per\nguidelines and in response to further reviewer’s critique. We hope you find the revised\nmanuscript to be acceptable for publication at Nature Communications. Our response to\nreviewer’s comment is as follows. Modifications in response to reviewer’s comment are\nhighlighted in blue.\nComment: “To explain the Nb20’s anti-depression effect, authors proposed a model: Binding of\nNb20 to the extracellular ligand binding domain of mGlyR promotes specific stabilized\nconformation of the receptor which propagates through the stalk region to remodel the 7TM\nregion, which then serve as the contact site for RGS7. Authors provided a detailed description in\nthe text; ““…remodeling at the 7TM interface triggers changes in contacting residues of the RGS\ncomplex. Nb20 binding appeared to shift CTH2 by 2.2 Å, induce a 1.2 Å displacement in RGS7,\nand 1.5 Å movement in the TM3-TM4 connecting loop, and up to a 5.5 Å (when compared with\n7SHF) changes in the TM3, which serve as the contact site for RGS7...””\nIn previous review, I have commented that comparison of the structures of the 7TMs between the\nNb20-bound- and Nb20-free mGlyR-RGS7/Gβ5 (PDB ID: 7EWP) showed less than 1 A\ndifferences in Ca atoms. While authors agreed with my observation on small structural difference\nin 7TMs, they maintained their claim that the 7TMs has been remodeled by Nb20-binidng, and\nretained their description in P9 in the main-text. I am not convinced on authors’ claim. I\nunderstand that the authors tried to explain structural differences using their previously published\nstructure alone (PDB ID: 7SHF). But there is other structure available which should be also used\nfor comparison. This is very simple issue. There is Nb20-free mGlyR-RGS7/Gβ5 structure\nwhich is determined independently and has been available in database for several years.\nComparison with this structure with the current Nb20-bound mGlyR is unbiased. Its 7TMs\nstructure is virtually identical to that of the Nb20-bound mGlyR-RGS7/Gβ5 presented in this\nwork. Can authors claim structural changes in 7TMs, although Nb20-binding does not affect the\nstructure of 7TMs of mGlyR? If so, how would the authors explain the structural\nidentity/similarity of 7TMs between them? Instead of maintaining their claims on conformational\nchanges in 7TMs, authors may consider other possibilities. In my opinion, authors can use Nb20-\nmGlyR interaction, their strong biological data with animal model, which would make more\nclear story.”\nResponse: We agree with the reviewer that the remaining issue is simple and essentially deals\nwith the interpretation. In our opinion, structures present ground truth. Extensive review of our\nwork has not identified any issues with the quality of the structure that we present. Thus, the\nstructural information presented in this manuscript can be analyzed by anyone and interpreted in\nmultiple possible ways. In this manuscript, we provide our analysis and interpretations. We have\ninjected significant transparency during the last revision comparing our Nb20 structure with the\napo structure we obtained earlier 7SHF and also the one that reviewer favors – 7EWP. The\nformer comparison shows more extensive changes, the latter less. In our opinion, comparisons of\nthe structures determined by the same group using identical conditions for structural work is\nmore accurate than across groups as conditions, e.g. choice of detergent, etc can influence the\nstructure. With this, the differences in opinion become an interpretational issue and as authors we\nwould like to be able to voice our opinion on the matter.\nNevertheless, we understand reviewer’s point and the editorial view and in response we have\nprovided further clarifications and acknowledgement of limitations and possible interpretational\ndifferences. Specifically, we adjusted the description of our model on pages 9 and 10 to suppress\nconclusions about changes in 7TM architecture caused by Nb20 binding. We also acknowledged\ngaps and limitations very explicitly in the Discussion on page 15 stating the following. “How\nexactly Nb20 influences the conformation of 7TM region and particularly its interface with\nRGS7/Gb5 has not been fully resolved in this study, given the differences between two previously\nreported ground state structures of mGlyR without Nb20. Additional structural work that\ncaptures intermediate conformations of the receptor complexes will be required to fully resolve\nthe mechanism of RGS/Gb5 modulation by mGlyR ligands such as Nb20 or glycine.”\nThank you for your consideration!\nBest wishes,\nKirill Martemyanov"}]

Decision letter