Molecular basis of antagonism of the dimeric human arginine vasopressin receptor 1A

Domain: biology
Published: 2026-01-16
Editorial outcome: major_revision

Abstract
Arginine vasopressin (AVP) and oxytocin (OT) are peptide hormones critical for various physiological processes. Vasopressin receptor 1A (V1aR), a primary AVP target, is promising for central nervous system (CNS) disorder therapies, yet the mechanisms of antagonism and oligomerization remain poorly understood. Here, we present structures of human V1aR in its apo state and in complexes with antagonists: atosiban, balovaptan, and SRX246. Structural analyses reveal a dimeric V1aR assembly, validated by functional assays and imaging in cells. The apo structure shows a flat extracellular loop 2 (ECL2) with unpaired cysteines, undergoing significant conformational changes upon ligand binding. Antagonist-bound structures, combined with mutagenesis and radioligand binding assays, uncover distinct binding modes and key determinants for antagonism and selectivity. These findings provide a comprehensive understanding of V1aR assembly and dynamic regulation, offering valuable insights for structure-guided development of new antagonists targeting dimeric V1aR for CNS disorders.

Introduction
Arginine vasopressin (AVP) and oxytocin (OT) are structurally homologous neuropeptides that regulate a variety of physiological processes through the AVP/OT receptor family: oxytocin receptor (OTR) and three vasopressin receptors (V1aR, V1bR, V2R)1–3. Among these, the vasopressin V1a receptor (V1aR), encoded by AVPR1A, is notable for its broad tissue distribution in both peripheral tissues and the central nervous system (CNS)2,4,5. Mechanistically, V1aR primarily couples with Gq/11 proteins, activating calcium-dependent signaling and protein kinase C (PKC) pathways6–8. These signaling pathways support the canonical roles of V1aR in cardiovascular and renal homeostasis, while also modulating higher-order CNS functions, including social behavior9–11, emotional reactivity12, stress adaptation13,14, and circadian timing15–17. Emerging evidence has implicated the dysregulation of V1aR signaling in several neurodevelopmental and neuropsychiatric disorders, such as autism spectrum disorder (ASD)18–20, post-traumatic stress disorder (PTSD)21,22, and Huntington’s disease (HD)23,24. This highlights V1aR as a promising therapeutic target for CNS disorders.

In line with its clinical relevance, several V1aR antagonists have progressed to various stages of therapeutic development5. Atosiban, a cyclic peptide derivative with dual inhibitory properties for AVP and OT, is approved and commonly used in Europe for the treatment of preterm labor25. Additional antagonists, such as conivaptan (a dual V1aR and V2R antagonist), have been developed for the treatment of hyponatremia26. Furthermore, CNS-penetrant V1aR-selective antagonists, including balovaptan for autism spectrum disorder (ASD)27, and SRX246 for Huntington’s disease (HD)28, have demonstrated preliminary efficacy in early-phase clinical trials. Despite these promising pharmacological profiles, the molecular mechanisms governing antagonist binding and receptor subtype selectivity remain poorly understood. This knowledge gap is largely attributed to the absence of high-resolution structural data for V1aR and its complexes with various antagonists, which hinders the optimization of V1aR-specific drug candidates.

Several structures of AVP/OT receptor family members have been resolved recently, most of which represent active-state and inactive-state conformations of V2R and OTR29–36. These structures have provided valuable insights into the ligand-binding and receptor-activation mechanisms. However, no structural information is currently available for V1aR. Notably, all currently resolved structures of V2R and OTR depict monomeric receptor states, whereas substantial evidence suggests that AVP/OT receptor family members can form homo- or heterodimers, which play important roles in downstream signaling and trafficking37,38. These findings highlight the need for further structural investigations to elucidate the molecular mechanisms underlying receptor dimerization.

In this work, we present the high-resolution cryo-electron microscopy (cryo-EM) structures of human V1aR in its apo state and in complexes with three clinically relevant antagonists: atosiban, balovaptan, and SRX246. Our structural and cell-based analyses reveal the mechanistic details of V1aR homodimerization. Furthermore, the V1aR apo structure uncovers a “flat” extracellular loop 2 (ECL2) with unpaired cysteines, which undergoes significant conformational changes upon ligand binding. Comparisons among the three antagonist-bound structures highlight the key molecular determinants influencing ligand binding, receptor antagonism, and subtype selectivity. The functional significance of these residues is validated through site-directed mutagenesis and radioligand binding assays. Collectively, our findings provide a comprehensive mechanistic framework for understanding V1aR regulation and offer valuable insights for guiding drug development efforts targeting the dimeric V1aR in CNS disorders.

Results
The overall structure of human V1aR
To obtain the structure of human V1aR, we constructed an expression vector containing full-length wild-type human V1aR with a signal peptide followed by a FLAG tag at its N-terminus. However, the protein aggregated heavily upon purification using anti-FLAG resin under various detergent conditions. To improve the biochemical behaviors of the proteins, we truncated part of the unstructured N-terminus of V1aR and introduced two mutations, S341C and N344K, which are a subset of the eight mutations used previously to stabilize OTR29 for structural studies. The resulting construct, named V1aR_cryo, was purified to homogeneity using Lauryl Maltose Neopentyl Glycol (LMNG) and Cholesteryl hemisuccinate (CHS) as detergents. V1aR_cryo in its apo state eluted as a monodisperse peak on size-exclusion chromatography (Supplementary Fig. 1a), which made it a suitable construct for cryo-EM analysis. The SDS-PAGE gel showed two bands, which likely correspond to the monomeric and the SDS-resistant dimeric species.

To evaluate the functionality of the construct, we performed cell-based ligand-binding assays to compare the ligand-binding properties of V1aR_cryo with those of the wild-type receptor. V1aR_cryo expressed at a similar level to the wild-type protein (Supplementary Fig. 2c). Although the N344K mutation renders V1aR signaling-incompetent39, ligand binding property is mostly preserved. AVP binding to V1aR_cryo was modestly compromised, similar to the effects of equivalent mutations on OT binding to OTR29 (Supplementary Fig. 2a and Supplementary Table 3). Notably, V1aR_cryo remains highly sensitive to antagonists atosiban, balovaptan, and SRX246, enabling investigation of their molecular mechanisms, which are the focus of the current study (Supplementary Fig. 2b and Supplementary Table 3).

Next, we prepared V1aR_cryo in its apo form (V1aR_apo) and complex with atosiban (V1aR_atosiban), balovaptan (V1aR_balovaptan), and SRX246 (V1aR_SRX246) for cryo-EM analysis (Supplementary Fig. 1a, c–e). The initial map of V1aR_cryo was of very limited resolution (data not shown), probably due to a lack of sufficiently large features outside the micelle for accurate particle alignment. To address this problem, we performed in vitro nanobody screening (details are provided in Supplementary Information) and obtained one hit Nb1 after extensive validations (Supplementary Table 2). Nanobody Nb1 was able to form complexes with V1aR_cryo in its apo as well as ligand-bound states (Supplementary Fig. 1b–e).

We then subjected these four samples to single-particle cryo-EM analysis and determined high-resolution structures at an overall resolution of 2.6-3.0 Å assessed by the gold standard of Fourier shell correlation (FSC) = 0.143 (Fig. 1, Supplementary Figs. 3 and 4, and Supplementary Table 1). The high-quality maps for all four structures allow for reliable model building of the majority parts of V1aR, including all transmembrane (TM) regions as well as extracellular loops (ECLs). Decent local densities also facilitated accurate docking of ligands and placement of cholesterol molecules, as well as the assignment of water molecules in the structure of V1aR_SRX246 (Fig. 1 and Supplementary Fig. 5). The flexible N-terminal and C-terminal region of V1aR and some cytosolic loops between TMs such as intracellular loop 2 (ICL2) were not modeled due to maps not resolved very well in those regions. Although Nb1 was not resolved well in the final high-resolution reconstructions, the 6 Å low-pass-filtered map reveals extra densities adjacent to the cytosolic face of V1aR that closely match the shape of Nb1 (Supplementary Fig. 5e), indicating that Nb1 engages a conformationally heterogeneous epitope on the intracellular side of the receptor. We therefore infer that Nb1 serves as a fiducial mask sufficient to enable early particle classifications and global low-resolution alignment. However, its flexible orientation relative to the receptor leads to partial signal averaging and eventual loss of nanobody density during high-resolution refinement.

The dimer architecture
Previous studies have demonstrated that V1aR can form homodimers or heterodimers with related receptors such as V2R and OTR37,38. Bioluminescence resonance energy transfer (BRET) analyses have shown that these dimeric arrangements remain stable regardless of agonist or antagonist treatment, suggesting that ligand binding does not significantly alter the dimerization state38. These insights have also facilitated the development of a dimeric agonist for OTR40. Unlike the previously reported monomeric structures of other receptors in this family, our cryo-EM structures reveal V1aR in a dimeric form, observed in both the apo state and antagonist-bound states (Fig. 1). We also observed a small population of particles with a smaller diameter in raw images, likely corresponding to the monomeric species, and tried to select these during particle picking. However, due to their small size and low abundance, these particles were lost during subsequent data processing. In contrast, no clear trimeric or tetrameric particles were detected in our cryo-EM datasets-unlike CXCR4, for which recent cryo-EM studies have visualized trimeric and tetrameric assemblies41.

The dimer interface of V1aR is quite extensive, primarily mediated by TM1, with additional contributions from the extracellular region of TM2 and the cytosolic region of TM7 (Fig. 2a). A comparison of the four structures shows that the dimers of V1aR_apo, V1aR_balovaptan, and V1aR_SRX246 share highly similar overall architectures. However, the two copies of V1aR_atosiban adopt a slightly different angle within the membrane compared to the others (Supplementary Fig. 6a), suggesting relative plasticity of the dimer interface.

To independently investigate the oligomeric state of V1aR in live cells, which has been previously reported as dimers, we employed a single-molecule photobleaching (smPB) assay, a well-established method for probing protein oligomerization42,43. In this approach, mStayGold was fused to the C-terminus of wild-type V1aR (Fig. 2b). The resulting fusion construct behaved similarly to wild-type V1aR in the NFAT-driven luciferase assay under both agonist stimulation with AVP and antagonist-mediated suppression (Supplementary Fig. 7 and Supplementary Table 4). The fusion construct was expressed at very low levels by transient transfection for a short period of time in U-2OS cells, which lack endogenous V1aR expression. Maintaining low expression levels was essential to ensure assay reliability and minimize potential dimerization artifacts caused by overexpression. Using total internal reflection fluorescence (TIRF) microscopy with single-molecule sensitivity, exogenously expressed V1aR-mStayGold constructs appeared as discrete fluorescent puncta on the cell surface (Fig. 2c). Continuous low-intensity laser illumination induced stepwise photobleaching of mStayGold fluorescence (Fig. 2d). For each selected punctum (Fig. 2c), the number of photobleaching steps was quantified to determine the oligomeric state of the receptor (details are described in Supplementary Information). For wild-type V1aR, approximately 75% of analyzed puncta exhibited two-step photobleaching, while the remaining 25% showed single-step bleaching (Fig. 2e). These findings are consistent with the structural observations demonstrating that V1aR forms a homodimer, and previous reports.

The detailed interactions at the dimer interface involve polar interactions on the extracellular side and hydrophobic interactions within the TM region (Fig. 2g). For polar interactions, K521.33 (numbers in superscripts correspond to Ballesteros-Weinstein numbering)44,45 form hydrogen bonds with E481.29 on the opposing protomer, a configuration that is nearly identical in V1aR_balovaptan and V1aR_SRX246, but slightly altered in V1aR_atosiban concerning bond angles. In V1aR_apo, K521.33 forms hydrogen bonds with the backbone carbonyl oxygens of D1122.64 and I1132.65 from the other receptor copy, while also interacting with Y1152.67 from the other copy via a cation-π interaction. The hydrophobic interactions are formed by Cβ-Cβ, Cβ-CA, or Cβ-O side-chain contacts through a network of small hydrophobic residues A561.37/A591.40/V601.41/A631.44 across all four structures, with an additional contribution from V641.45 in V1aR_atosiban. Similar dimer interfaces, stabilized by residues with small side chains, have been observed in the dimer of Ste2, a class D GPCR46,47. To further validate our structural models, we introduced mutations at key interface residues. Specifically, K521.33 was mutated to alanine, and A591.40 was replaced with tyrosine, a residue with a bulky side chain. These mutations resulted in a mutant V1aR that predominantly exhibits a monomeric state on the cell surface (Fig. 2f). In the NFAT-driven luciferase assay, these mutations resulted in a higher EC50 value for AVP and higher IC50 values for all antagonists tested, which suggest that the dimer assembly is physiologically relevant (Supplementary Fig. 7 and Supplementary Table 4).

A comparative survey of reported class A GPCR homodimers further contextualizes the V1aR interface. The β1-adrenergic receptor (β1AR) and rhodopsin adopt a V1aR-like arrangement with a TM1-centered interface, whereas the principal contact surface is on TM5-TM6 in CXCR4, on TM5–TM6 in GPR3, and within ECL1 between TM2 and TM3 in APJR (Supplementary Fig. 8)48–56. Except for APJR, whose dimerization relies mainly on ECL1 contacts, these dimers including V1aR are stabilized predominantly by extended hydrophobic interactions within the TM region.

The ligand-free state
ECL2 in GPCRs is well-known for its length variation and structural diversity, playing crucial roles in receptor signaling57–59. Our structures reveal that ECL2 of V1aR adopts distinct configurations in the apo state compared to the antagonist-bound states (Fig. 3a), whereas the ECL2 conformations in the antagonist-bound states are very similar (Supplementary Fig. 6b). In the antagonist-bound states, ECL2 adopts an “up” conformation, forming an antiparallel β-sheet fold that projects away from the binding pocket at an approximately perpendicular orientation relative to the membrane (Fig. 3a). This “up” conformation of ECL2 creates a large, solvent-exposed cavity capable of accommodating ligands. In the apo state, the majority of ECL2 was modeled reliably, supported by well-defined local densities (Fig. 3b and Supplementary Fig. 6c). Notably, two segments that form the antiparallel β-sheet fold observed in the antagonist-bound states adopt alpha-helical conformation in the apo structure: one becomes a continuation of TM4, while the other inserts into the ligand-binding pocket (Fig. 3a and Supplementary Fig. 6c). The remainder of ECL2 (residues 193–201, sequence between the two beta-sheet segments) is visible as continuous density in the 6 Å low-pass-filtered map (Supplementary Fig. 5f), but could not be confidently modeled. Given the relatively planar orientation of ECL2 relative to the membrane in the apo structure, we designate this arrangement the “flat” conformation.

These extensive conformational changes in ECL2 are also accompanied by significant structural rearrangements in ECL1, the extracellular loop connecting TM2 and TM3 (Fig. 3a). One notable outcome of the conformational changes of ECL2 is their impact on the conserved disulfide bond between ECL2 and TM3, a hallmark feature of class A GPCRs57,60. In the antagonist-bound structures of V1aR, C203 in ECL2 forms a disulfide bond with C1243.25 in TM3. Interestingly, in the apo state, this canonical disulfide bond is not formed, with C203ECL2 and C1243.25 remaining unpaired (Fig. 3c). The cryoEM densities for both unpaired cysteines are well resolved and supported by clear density for surrounding residues (Fig. 3b).

Mechanistically, the inserted part of ECL2 in the apo structure is stabilized in the “flat” configuration through a combination of hydrophobic interactions and hydrogen bonds with residues from TM2, TM3, TM4, TM5, and TM6 (Fig. 3d). Specifically, W204ECL2 inserts into a hydrophobic pocket created by P1072.59, W1112.63, and K1283.29, while W2115.34 inserts into a separate hydrophobic pocket composed of Y2165.39, V2175.40, and M2205.43 on TM5, and F3076.51 on TM6. Additionally, stabilizing hydrogen bonds are observed between R118ECL1 and the carbonyl oxygen of D202ECL2, Q1082.60, and the backbone nitrogen of A205ECL2, K1283.29 and the carbonyl oxygen of W204ECL2, together with the side chain of Q209ECL2, Q1854.61 and the carbonyl oxygen of I208ECL2 (Fig. 3d). Collectively, these interactions anchor ECL2 within the binding pocket in the apo state. This inserted ECL2 with unpaired cysteines was also observed in the recently reported GPR4 structure61.

The antagonistic mechanism of atosiban
The cryo-EM structure of the V1aR_atosiban complex reveals that atosiban spans the entire ligand-binding pocket of V1aR. The high-quality density map enables precise modeling of various parts of the ligand (Fig. 4a). As an OTR/V1aR dual antagonist62,63, atosiban retains the nine-residue peptide backbone of oxytocin, the endogenous OTR agonist, including the conserved disulfide-bridged ring. However, it incorporates modifications at four key positions to confer proteolytic resistance and enhance pharmacokinetic properties: Cys1 is replaced with mercaptopropionic acid (Mpa), eliminating the free amino group; Tyr2 is replaced by D-Tyr(Et), a D-enantiomer bearing an ethoxy moiety; Gln4 is substituted with Thr; and Leu8 is replaced with Orn (Fig. 4e)64. In the cryo-EM structure, atosiban adopts a similar overall conformation to oxytocin (Fig. 4e) and engages V1aR through a highly coordinated network of polar and hydrophobic interactions that collectively stabilize the receptor in an inactive conformation (Fig. 4b). Mpa1 of atosiban forms a disulfide bond with Cys6 of atosiban, reinforcing the rigidity of the cyclic ring, and establishes a hydrogen bond with Q3116.55 of V1aR via its terminal carboxyl group. D-Tyr(Et)2 of atosiban inserts into a polar sub-pocket formed by TM1, TM2, and TM7, engaging Q1042.56 and S3387.42 through its ethoxy oxygen, while its terminal carboxyl group interacts with K1283.29 and Q1854.61 of V1aR (Fig. 4c). Notably, K1283.29 and Q1854.61 are essential residues for AVP recognition35. Due to their functional importance, mutations at these sites significantly impair [3H]AVP binding, making them unsuitable for use in competition assays involving atosiban, balovaptan, and SRX246. Mutational analyses highlight the important role of S3387.42, as mutations of this residue result in a 24-fold reduction in Ki (Fig. 4d, Supplementary Figs. 9 and 10a, and Supplementary Table 3). Other residues also contribute to ligand stabilization. Ile3 of atosiban is embedded in a hydrophobic pocket formed by TM3, TM5, and TM6, contacting M1353.36, Y2165.39, V2175.40, M2205.43, and F3086.52 of V1aR (Fig. 4c). Mutagenesis of these positions (V217A, M220I, F308A) significantly reduces atosiban binding, highlighting the functional importance of this hydrophobic cleft (Fig. 4d). Thr4 of atosiban forms a stabilizing hydrogen bond with Q3116.55 of V1aR, while Asn5 of atosiban contributes to ligand recognition via a hydrogen bond with backbone oxygen of A205ECL2 and amino-π stacking with W204ECL2 of V1aR. The linear tail of atosiban—comprising Pro7, Orn8, and Gly9—extends towards the extracellular vestibule. Orn8 further stabilizes the ligand by forming hydrogen bonds with E541.35 and T3317.35 of V1aR, reinforcing the anchorage of the C-terminal segment.

In the absence of the V1aR-AVP complex for direct comparison, we compared the structures of V1aR–atosiban and OTR–oxytocin complexes (PDB: 7RYC)33 to gain deeper insights into ligand recognition and receptor antagonism. The ligand-binding poses in the two complexes exhibit a high degree of overlap (Fig. 4f). However, distinct ligand binding results in noticeable differences between the two receptors, particularly in the extracellular portion of TM1, the intracellular portions of TM5, as well as TM6 and TM7 (Fig. 4g). A key difference lies in the remodeling of TM7. In the OTR–oxytocin complex, Tyr2 of oxytocin forms a critical hydrogen bond with the carbonyl oxygen of L3167.39 and induces a bend in TM7 near A3187.41, promoting rearrangement of W2886.48 on TM6 and activating the CWxP toggle switch, a key structural hallmark of GPCR activation65–68. In contrast, the substitution of Tyr2 with D-Tyr(Et) in atosiban eliminates the phenolic hydroxyl group, in the meantime, the additional ethyl group on D-Tyr(Et)2 enhances local hydrophobicity and shape complementarity, which collectively help maintaining TM7 in its canonical α-helical form, thus stabilize the receptor in its inactive state (Fig. 4h). Meanwhile, in the structure of OTR–oxytocin complex, F2846.44, a key residue within the conserved PXF motif of class A GPCRs, undergoes an outward movement induced by the aforementioned conformational rearrangement of W2886.48, thereby promoting OTR activation. In contrast, in the V1aR–atosiban complex, W3046.48 retains its canonical inactive conformation, preventing the conformational transition of Y3006.44 (the equivalent of F in the PXF motif) that is required for V1aR activation (Fig. 4j).

Additionally, in the V1aR-atosiban structure, an ionic lock involving R1493.50, D1483.49 in DRY motif69, and T2896.33 stabilizes TM6 in an inward conformation, preventing the formation of the G protein–binding cavity. In contrast, in the active OTR–oxytocin complex, this ionic lock is disrupted: R1373.50 disengages from D1363.49 and instead stacks with Y243 of the miniGq/i, thereby stabilizing the active conformation (Fig. 4k). Consequently, in the V1aR-atosiban structure, the cytoplasmic end of TM6 fail to undergo the outward displacement required for G protein engagement, rendering the intracellular cavity inaccessible to the α5 helix of Gαq. The tail of atosiban (Pro7, Orn8, and Gly9) also contributes to the inactivation of V1aR. Previous studies have revealed the important roles of Mg2+ in the activation of both OTR and V1aR29,33. In the V1aR-atosiban structure, the binding of the atosiban tail positions Orn8 very close to where Mg2+ is located in the OTR–oxytocin structure. This, combined with the altered local structure, results in the exclusion of Mg2+ (Fig. 4i).

Besides the OTR–oxytocin active-state structure, V2R–AVP signaling complexes have also been resolved and provide family-level templates for comparison. Consistent with pharmacological profiles, V1aR binds AVP with markedly higher affinity than oxytocin70. To gain insights into this difference, we computationally generated active-state models of V1aR–AVP and V1aR–oxytocin using MODELLER71,72, followed by restrained docking, guided by the OTR–oxytocin and V2R–AVP structures (Supplementary Fig. 11)32,33,72. Because AVP and oxytocin differ only at positions 3 and 8 (AVP: Phe3/Arg8; oxytocin: Ile3/Leu8), our models attribute the selectivity to two cooperative features: (i) Phe3 (AVP) inserts more deeply into the TM5–TM6 hydrophobic cleft than Ile3 (oxytocin), enhancing hydrophobic interactions. Moreover, Met2005.43 of V1aR will clash with Ile3 (oxytocin), whereas in OTR and V2R, isoleucine at this position can accommodate Ile3 (oxytocin). (ii) Arg8 (AVP) forms electrostatic contacts with residues in TM1, stabilizing the rim interactions.

Together, these features elucidate the molecular mechanism by which atosiban stabilizes V1aR in an inactive state, effectively preventing G protein engagement and downstream signaling.

Binding modes of balovaptan and SRX246
In addition to the peptide-based antagonist atosiban, we accurately resolved the binding modes of two representative V1aR-specific small-molecule antagonists: balovaptan and SRX2465. Both small molecules antagonize the V1aR receptor by inducing a conformation change very similar to that caused by atosiban (Supplementary Fig. 6b). While both occupy the orthosteric pocket (Fig. 5a, b), balovaptan and SRX246 exhibit distinct chemotypes and binding modes.

Balovaptan is a non-peptidic small-molecule V1aR antagonist, originally developed by Roche for the treatment of autism spectrum disorder (ASD), aiming to modulate AVP-mediated social cognition pathways27. Balovaptan features a rigid tricyclic core composed of a [1,4]-benzodiazepine fused with a [1,2,4]-triazole ring. This core scaffold is further decorated with a methyl group at position 5 and a chlorine substituent at position 8, which enhance the hydrophobic character of the molecule (Fig. 5c). These hydrophobic features enable balovaptan to deeply insert into the ligand-binding pocket of V1aR (Fig. 5a), which is lined by V1323.33, M1353.36, Y2165.39, V2175.40, M2205.43, F3076.51, and F3086.52 (Fig. 5d). Mutagenesis of key residues within this pocket impairs ligand binding to varying degrees, with V217A showing the most significant effect, underscoring the essential role of this residue in stabilizing the molecule (Fig. 5g, Supplementary Figs. 9, 10b, and Supplementary Table 3). Additionally, the triazole nitrogen atoms form polar contacts with K1283.29 and Q1854.61, further stabilizing the core scaffold within the pocket. Extending from its tricyclic core, balovaptan possesses a flexible cyclohexyl–ether–pyridyl side chain that projects towards the polar sub-pocket formed by TM1, TM2, and TM7. The ether oxygen forms a hydrogen bond with Q1042.56, while the pyridine nitrogen engages in a polar network involving S3387.42 and Q1042.56 (Fig. 5d). These interactions are further supported by mutational data (Fig. 5g). Notably, mutation of A3347.38 on TM7 to methionine—the equivalent residue found in V1bR, V2R, and OTR—introduces a potential steric clash with the pyridyl ring, resulting in a decrease in binding affinity (Fig. 5g). This observation indicates the importance of steric compatibility at the pocket entrance for optimal ligand accommodation and partially explains the receptor subtype specificity for balovaptan.

SRX246, developed by Azevan Pharmaceuticals, is another non-peptidic V1aR antagonist currently under clinical investigation for the treatment of post-traumatic stress disorder (PTSD)73 and other affective disorders, such as HD28,74, which are linked to AVP signaling. Unlike balovaptan, SRX246 employs a structurally modular strategy centered on a rigid β-lactam (azetidinone) core, flanked by four discrete functional regions (Zones A–D)75,76 that engage distinct subpockets within the receptor through fragment-like interactions (Fig. 5b, e). Zone A consists of an oxazolidin-2-one linked to a terminal phenyl ring. The aromatic moiety is buried within a hydrophobic subpocket formed by F1894.65, F207ECL2, and Y2165.39, while the carbonyl group of the oxazolidinone engages in water-mediated hydrogen bonds with K1283.29 and Q1854.61. K1283.29 also forms a hydrogen bond with the carbonyl group of the azetidinone core directly (Fig. 5f). Zone B contains a styrenyl-like π-conjugated system co-planar with the β-lactam ring, reinforcing molecular rigidity and π–π interaction potential. The terminal phenyl ring inserts into the same but deeper hydrophobic cleft between TM3 and TM6, forming extensive contacts with M1353.36, V2175.40, M2205.43, F3076.51, and F3086.52 (Fig. 5f). Functional mutagenesis further validates the structural dependency of Zone B, as mutations such as V217A, M220I, and F307Y significantly reduce binding affinity (Fig. 5h, Supplementary Figs. 9 and 10c, and Supplementary Table 3). Notably, the equivalent residue of M2205.43 in V2R and OTR is isoleucine, which partially explains the receptor subtype specificity of SRX246. Zone C consists of a phenylethyl group linked to the central amide nitrogen. The phenylethyl group is attached via an amide linkage, with its phenyl ring forming a polar interaction with Q1042.56. Meanwhile, the carbonyl oxygen of the amide engages in a hydrogen bond with K1283.29. Zone D extends into the extracellular vestibule through a bipiperidine group that forms a polar interaction with E541.35 and engages in hydrophobic interaction with A511.32 (Fig. 5f), further stabilizing the distal portion of the molecule and contributing to the overall rigidity of the ligand–receptor complex.

Collectively, SRX246 exemplifies a fragment-based multivalent design, featuring a rigid central core paired with adaptable peripheral regions to achieve comprehensive engagement of the orthosteric site. In contrast, balovaptan employs a single deep-binding hydrophobic core, complemented by a flexible polar extension that interacts with the extracellular face of the receptor. In both antagonist-bound structures, Mg2+ was not observed due to the disruption of the Mg2+ binding site. Despite their contrasting structural architectures, both ligands stabilize V1aR in a conformation that is incompatible with G protein engagement, underscoring distinct yet convergent strategies for receptor antagonism.

Comparison of AVP/OT family-antagonist interactions
We first observed that SRX246 exhibits a modular architecture, with distinct chemical moieties occupying spatially separable regions of the V1aR binding pocket. Leveraging this feature, we used SRX246 as a reference scaffold to compare antagonist binding modes across receptors in the AVP/OT family29,35. For clarity, substructures extending into zones A and D were collectively assigned to the “upper pocket”. The “upper pocket” comprises the extracellular-facing portion of the orthosteric cleft, including the interface with the extracellular loops (ECLs). In contrast, moieties corresponding to zones B and C were assigned to the “bottom pocket” that is the deeper transmembrane cavity (Fig. 6a).

We then aligned available antagonist–receptor complex structures to the V1aR–SRX246 complex to compare ligand binding poses. This analysis revealed that AVP/OT-family antagonists can be broadly grouped into three classes based on their penetration depth and extent of pocket engagement. Class I is represented by atosiban, a macrocyclic nonapeptide antagonist that, like SRX246, spans nearly the full width and depth of the orthosteric site—engaging both the upper and bottom pockets. It further extends toward the pocket mouth of the binding pocket to make auxiliary contacts with polar residues on TM1. Class II is exemplified by mambaquaretin-1 (MQ1), a compact, toxin–like mini-protein antagonist. In contrast to SRX246, MQ1 primarily occupies the upper pocket and forms extensive contacts with the ECLs, with limited insertion into the deeper transmembrane cavity. Class III consists of small-molecule antagonists such as retosiban, balovaptan, and tolvaptan. These ligands are enriched in hydrophobic and heteroaromatic ring systems and preferentially occupy the bottom pocket. Unlike peptide- or protein-like antagonists, these compounds make limited contact with the ECLs and instead exploit the hydrophobic subcavity at the base of the orthosteric site.

Methods
Recombinant expression and purification of V1aR
The original sequence of human V1aR (Uniprot: P37288) (residues 33–418) with S341C and N344K mutations, and an N-terminal signal peptide followed by a FLAG tag and Avitag, was cloned into a pEG-BacMam vector1. The correct plasmids were transformed into DH10Bac for bacmid production. Validated bacmids were transfected into 1.5×106 Sf9 cells (Thermo Fisher Scientific, #11496015) using Cellfectin™ II Reagent (Thermo Fisher Scientific) for virus production. Subsequently, 33 mL of P2 virus was added to 1 L of Expi293F cells (Thermo Fisher Scientific, #A14527) maintained at a density of 2.8–3×106/mL at 37°C in SMM 293-TII expression medium (Sino Biological) supplemented with 1% fetal bovine serum (FBS, CellMax). After 15-16 hours of transduction, sodium butyrate was added to a final concentration of 10 mM to enhance protein expression. The cells were cultured for an additional 72 hours at 30°C before being harvested by centrifugation (2500×g, 15 min).

The cells were sonicated at 50% strength (2s on/3s off) for a total of 10 min in a buffer containing 25 mM HEPES pH 7.4, 300 mM NaCl, and 0.5 mM PMSF. After cellular debris was removed by centrifugation at 8000×g for 15 min, the supernatant was subjected to ultracentrifugation (43,000 rpm for 1.5 h at 4°C) to pellet the membrane. The membrane pellet was resuspended and homogenized using a douncer in buffer (25 mM HEPES pH 7.4, 300 mM NaCl supplemented with protease inhibitor cocktail). Different antagonists were added to the membrane suspension at indicated final concentrations (5 μM atosiban/5 μM balovaptan/4 μM SRX246) and incubated at room temperature for 30 min to allow binding. The membrane pellet for the apo state was used directly for downstream purification.

The membrane was solubilized in a Buffer (25 mM HEPES pH 7.4, 300 mM NaCl, 1% (w/v) LMNG (Anatrace), and 0.1% (w/v) CHS (Anatrace)), supplemented with protease inhibitor cocktail and with or without the corresponding antagonists, for 1.5 hours at 4°C. The insoluble materials were removed by ultracentrifugation (43,000 rpm for 1.5 h at 4°C), and the supernatant was incubated with anti-DYKDDDDK G1 affinity resin (GenScript) for 2.5 h at 4°C. The resin was washed with washing buffer (25 mM HEPES pH 7.4, 300 mM NaCl, 0.005% (w/v) LMNG, and 0.0005% (w/v) CHS, with/without the corresponding antagonists), for 10 column volumes, including an ATP wash step using buffer supplemented with 2 mM ATP/5 mM MgCl2. The protein was then eluted with the same washing buffer supplemented with 0.3 mg/mL 3xFLAG peptide (Biotool). Eluted proteins were further purified by size-exclusion chromatography (SEC) using a Superose 6 Increase 10/300 GL column (Cytiva) in a buffer containing 25 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, 0.005% (w/v) LMNG, and 0.0005% (w/v) CHS with/without the corresponding antagonists. Peak fractions were collected and concentrated for subsequent experiments.

Selection, production, and characterization of nanobodies for V1aR
The purified and biotinylated V1aR was used for nanobody screening. The procedure followed a similar protocol including one round of ribosome display and two rounds of phage displays that were previously described77,78 and modified later79. The corresponding antagonist was included throughout the selection process. Individual positive clones were expressed and identified by ELISA followed by sequencing. Plasmids of desirable hits were transformed into E. coli BL21(DE3) competent cells and then expressed and purified similarly as previously described79. A pull-down assay was used to characterize the binding of nanobodies with V1aR in its apo state or bound with different antagonists. One hit (Nb1) that binds V1aR in all states was identified and used for the following cryo-EM analysis.

Assembly of V1aR_cryo/Nb1 complex
Purified V1aR_cryo in its apo state or bound with different antagonists was incubated with nanobody Nb1 at a molar ratio of 1:1.5 on ice for 1 hour. The complex was then purified on a Superdex 200 Increase 3.2/300 (Cytiva) column in a buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 0.01% LMNG, 0.003% GDN, and 0.001% CHS, with or without the corresponding antagonists. Peak fractions were pooled and concentrated for cryo-EM analysis.

Cryo-EM Sample Preparation and Data Collection
A total of 3 μL of the purified sample (5–6 mg/mL) was applied to glow-discharged (Coolglow, SuPro Instruments) holey carbon grids (Quantifoil R1.2/1.3 400 mesh). The grids were blotted for 4s at ~100% humidity and 4°C, and then plunge-frozen in liquid ethane using the Vitrobot Mark IV (Thermo Fisher Scientific). All cryo-EM data were collected on a Titan Krios G4 (FEI) operating at 300 kV, equipped with a Selectris X imaging filter (Thermo Fisher Scientific) and a Falcon 4i direct electron detector (Thermo Fisher Scientific). An imaging filter with a slit width of 10 eV was used. All cryo-EM movies were recorded using EPU (Thermo Fisher Scientific), with a total electron dose at 50 electrons/Å2. The nominal magnification of 130kx corresponds to a calibrated pixel size of 0.92 Å on the specimen. The defocus range for the samples was between 1.0 and 2.0 μm.

Image processing
The image processing workflows are illustrated in detail in Supplementary Fig. 3 and were performed using cryoSPARC v480. Briefly, motion correction and dose weighting were performed using the Patch_Motion_Correction module. After CTF was estimated with the Patch_CTF module, micrographs with CTF-estimated resolution worse than 3.5 Å, high drift values, and extreme defocus range were excluded from downstream data analysis.

Data analysis was conducted iteratively. The initial set of particles from approximately 200 micrographs was selected using Blob_Picker. Two-dimensional (2D) classifications were performed on these selected particles, and 2D averages that displayed clear protein features were used as initial templates for particle picking across all chosen micrographs with the Template_Picker module. After two rounds of 2D classifications to eliminate junk particles and false picks, particles corresponding to the good 2D class averages were chosen for an Ab-initio 3D reconstruction. Clear secondary structure features were readily apparent in the initial 3D reconstruction. This initial 3D volume was then employed to generate 40 2D templates using the Create_Templates module. These generated 2D templates once again served as templates for particle picking across all selected micrographs. After two rounds of 2D classifications, high-quality particles underwent one round of heterogeneous refinement using one “good” 3D volume and three “bad” 3D volumes. Non-uniform refinement was subsequently performed on the particles from the selected good class, followed by Local_CTF_refinement and Reference_based_motion_correction. The CTF-refined and polished particles were then subjected to another round of heterogeneous refinement using one or two “good” 3D volumes and two or three “bad” 3D volumes. Particles from the good class that exhibit clear secondary structural features and small molecule densities were used for the final round of Non-Uniform refinement.

Local resolution variations were estimated using Local_Resolution module. Resolutions of the refinement were estimated according to the gold-standard Fourier Shell Correlation (FSC) 0.143 criterion.

Model building and refinement
AlphaFold prediction81 of the human V1aR (AF-P37288-F1) was used as an initial model for model rebuilding into the cryo-EM density map of high resolution. The initial model was first manually docked into the cryo-EM density map using UCSF Chimera82. Iterative manual rebuilding and the addition of water molecules, antagonists, and cholesterol were performed in Coot83. Then, the models were refined using Phenix84. Structural figures were prepared using UCSF ChimeraX85.

Cell-based ligand-binding assay
Different V1aR constructs were transiently transfected into HEK293T cells (ATCC, CRL-3216) for the binding assay. 24 hours after transfection, cells were digested and re-plated at about 40% confluency on a poly-L-lysine-coated (0.1 mg/mL) (Sigma-Aldrich, #P4707) 48-well tissue culture plate one day before experiments. The expression level of V1aR constructs was analyzed by Western blot using an anti-HA antibody at 1:3000 dilution (Sigma, 3F10, #11867423001). On the day of the experiment, cells were counted and washed once with binding buffer (10 mM MgCl2 and 0.2% BSA in PBS) at room temperature. For the [3H]AVP saturation binding assay, increasing concentrations (indicated in the figure) of a radiolabeled AVP mixture ([3H]AVP (Revvity): unlabeled AVP (MCE) at 1:3) diluted in the binding buffer were added to initiate the binding, and the binding was carried out at room temperature for 30 min. Non-specific binding was determined similarly by diluting the radiolabeled AVP mixture in the binding buffer containing 10 μM unlabeled AVP. Specific binding for AVP was calculated by subtracting non-specific background binding from the total binding. To determine the IC50 values of antagonists, competition ligand-binding experiments were performed. Transfected and washed cells were incubated with increasing concentrations of antagonists together with 4 nM of radiolabeled AVP mixture ([3H]AVP: unlabeled AVP at 1:3) for 30 min on ice.

After the binding, cells were washed three times with the ice-cold binding buffer quickly. The cells were then lysed in 50 μL of binding buffer containing 1% Triton X-100 while shaken at 80 rpm at 37°C. The total lysate was then transferred to a tube containing a scintillating agent (PerkinElmer). Radioactivity was measured by MicroBeta2 Microplate Counter (PerkinElmer). Data was analyzed in GraphPad Prism 9 to obtain the Kd, Bmax, and IC50 values. Antagonist affinity was reported as Ki. For each antagonist, the IC50 from the competition curve (four-parameter logistic fit) was converted to Ki using the Cheng–Prusoff equation for competitive binding.

Luciferase reporter assays
HEK293T cells were seeded in 6-well plates and transfected at 70%-90% confluency using Lipofectamine 3000 Transfection Reagent (Vazyme, TL301), according to the manufacturer’s instructions. Each well received 1.2 μg of plasmid encoding the indicated receptor construct together with 1.2 μg of an NFAT-driven firefly luciferase reporter plasmid that generates NFAT response through the Gαq-dependent signaling. Approximately 8 h post-transfection, cells were digested and re-plated at about 40% confluency on a poly-L-lysine-coated (0.1 mg/mL) 48-well plate. Cells were allowed to adhere for 18 hours before compound treatment.

For agonist dose-response experiments (determination of EC50 value of AVP), cells were stimulated with serial dilutions of arginine vasopressin (AVP) at indicated concentrations and incubated for 5 h at 37°C. For antagonist inhibition assays (determination of IC50 value of antagonists), serial dilutions of the test antagonists were prepared and mixed with AVP at a fixed concentration (EC80, defined from the AVP concentration–response curve described above). The antagonist/AVP mixtures were added to the cells, which were then incubated for 5 h at 37°C.

After stimulation, cells were first washed once with DPBS and then lysed directly using Passive Lysis Buffer (Promega, E1910) for 15 min at room temperature with gentle shaking. Firefly luciferase activity was quantified using an Agilent BioTek Neo2 multimode plate reader according to the manufacturer’s instructions (Promega). For EC50 analyses, raw firefly luciferase signals were normalized to the vehicle-treated baseline and expressed as a percentage of maximal AVP response. For IC50 analyses, the signal obtained with AVP (concentration at EC80) in the absence of antagonist was defined as 100%, and all other signals were expressed as % of this control.

Concentration–response curves were fitted in GraphPad Prism (GraphPad Software) using a four-parameter logistic (variable-slope) model with log10[ligand] (agonist or antagonist) as the x-axis. Half-maximal effective (EC50) and inhibitory (IC50) concentrations were obtained from the fitted curves.

Single-molecule photobleaching assay
U-2 OS cells (ATCC, HTB-96) were maintained in complete growth medium comprising McCoy’s 5A (Gibco, #16600-082) supplemented with 10% fetal bovine serum (Sigma-Aldrich, #F8318) and 1% penicillin-streptomycin (Gibco, #15140122). High-precision coverslips (Marienfeld, #0117650) were cleaned by sequential treatment in 1 M KOH solution (Sigma-Aldrich, #484016) with 15 min of ultrasonic cleaning, followed by three 10-min rinses in ultrapure water. Sterilization was performed by immersion in ethanol (J&K Scientific, #258449) prior to coating with poly-L-lysine (Sigma-Aldrich, #P4707).

After 24 h of attachment to coverslips, U-2 OS cells were transfected separately with 1 μg of mStayGold-tagged wild-type (WT) or K52A/A59Y constructs using Lipofectamine™ 3000 (Thermo Fisher Scientific, #L3000015) according to the manufacturer’s instructions. At 3 h post-transfection, cells were fixed with 3% paraformaldehyde (PFA; Electron Microscopy Sciences, #15710) and 0.1% glutaraldehyde (GA; Electron Microscopy Sciences, #16019) in PBS (Gibco, #10010023) for 15 min at room temperature, followed by three rinses with PBS. Samples were mounted on slides with PBS prior to imaging.

Total internal reflection fluorescence (TIRF) imaging was performed on a custom-built microscope. The samples were excited with a 488 nm laser (Coherent OBIS 488LX, 150 mW). The mStayGold signal was collected using a TIRF objective (Olympus, 100×/1.5 NA, oil immersion) and detected by an EMCCD camera (Andor IXON-L-888) with a gain setting of 300. All data were acquired at 488 nm laser intensity of ~90 W/cm².

Raw image stacks comprising 3000 frames were acquired at a frame rate of 5 Hz. Two researchers independently selected approximately 100 non-overlapping, single-spot regions from each stack using ImageJ software. The summed fluorescence intensity within a 3×3-pixel region of interest (ROI), centered on each spot, was computed. Fluorescence intensity profiles across the entire stack were then plotted to identify photobleaching steps for individual spots. One researcher manually classified the bleaching curve types, and a second researcher performed an independent review. Disputed spots, including those showing more than two photobleaching steps or lacking clear, well-defined transitions in their intensity profiles, were excluded from further analysis. The number of spots exhibiting distinct bleaching steps was quantified for each experimental group and subjected to statistical analysis. Each group included three replicates derived from two independent experiments.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Zhong P et al. describes the cryo-electron microscopy (cryo-EM) three-dimensional (3D) structures of the\nhuman vasopressin V1a receptor (V1aR) in complex with three different types of antagonists, and in its apo state. These are\nthe first high-resolution structures of V1aR ever described. Interestingly, all structures presented in this manuscript are those\nof homodimeric V1aR particles. In addition, the authors show that the apo state structure of V1aR displays a flat extracellular\nloop 2 (ECL2), a significant difference in conformation with that of the antagonist-bound receptor states (in which this\nextracellular loop exhibits a typical antiparallel beta-sheet).\nThis work is a nice combination of recombinant receptor expression and purification, nanobody selection and production,\ncryo-EM structure acquisition and analysis, site-directed mutagenesis and cell-based pharmacological assays. This study\nprovides a comprehensive understanding of V1aR assembly and opens the way to structure-guided development of new\nantagonists for treating central nervous system disorders. It also gives information about antagonist receptor selectivity.\nThe methods developed are state-of-the-art and the results presented in the article are really interesting for G protein-\ncoupled receptor (GPCR) research community. However, I have one major concern and also some other criticisms before\nthe manuscript would be accepted for publication in Nature Communications.\n1/ major comment:\nMy main criticism is focused on the ligand-free state of the V1aR. The author claim that the structures reveal a distinct\nconformation of ECL2 in the apo state compared to the antagonist-bound states. I agree with this nice idea but I think the\npresent results do not support the conclusions.\nThe authors indicate that in the apo state, the ECL2 adopts a flat configuration whereas in the ligand-bound states this loop\nis in an open (up) conformation with a typical antiparallel beta-sheet conserved in most class A GPCRs. Moreover, the\ncanonical disulfide bond between ECL2 and transmembrane domain 3 (TM3) is absent in the apo state while it is present in\nthe antagonist-bound conformations. I think this disulfide bond is not seen because the ECL2 could not be constructed\ntotally. Why the conserved disulfide bond would not be formed in the apo state? If I remember well, this post-translational\nmodification should be done in the endoplasmic reticulum, before targeting the receptor to the plasma membrane and before\ninteractions with ligands. Again, the antiparallel beta-sheet is not seen since part of the loop is missing in the\ndensity map, and amino-acid residues 191-201 are not present in the corresponding model. ECL2 is probably more flexible in the ligand-\nfree state, thus I can understand it is more difficult to get a high-resolution density map in this region.\nHowever, would it be possible to collect more movies (and substantially increase the number of particles) with this apo state\nsample in order to be able to solve completely the ECL2 and to conclude accurately? Indeed, authors collected only 4860\nmovies for the apo-state, and were able to collect 12117 or 6163 for the atosiban-bound state and the balovaptan-bound\nstate, respectively.\nIf a new cryo-EM session is acquired and if the complete density of the ECL2 is seen, I would be happy with the authors\nconclusion. It would be quite interesting to discuss the role of the ECL2 in a “flat” conformation and its transfer into an “up”\nconformation in the presence of ligands. Could this loop close the solvent-exposed orthosteric binding pocket to help in\nmaintaining a basal receptor state? Could this loop mimic a neutral ligand (no activation)? Since this apo state is the first\none solved in the vasopressin/oxytocin receptor family as compared to active and inactive conformations of vasopressin V2\nreceptor (V2R) and oxytocin receptor (OTR) already available, the conclusions about the difference in ECL2 conformations\nbetween ligand-free and antagonist-bound states are crucial.\n2/ minor criticisms:\na) If we look at the density map of the apo state, there is a density on the intracellular side at the dimer interface between\nTM1 and TM7 which has not been assigned. This density is present in the two copies of the V1aR, symmetrically. Would this\ndensity correspond to a phospholipid? To a cholesterol molecule? Could this density correspond to part of helix 8 which is\nactually not seen?\nb) Again, in the apo state structure, if we overlap the density and the corresponding three-dimensional model at the level of\nthe extracellular loop 3 (ECL3), it is quite difficult to fit the residues in the density. I think some residues of the ECL3 should\nbe removed.\nc) All the structures solved in this study are those of dimeric V1aR. Indeed, size-exclusion chromatography (supplementary\nfigure 1) and SDS-PAGE of the cryo-EM samples show purified dimeric species, in agreement with the homodimers seen in\nlive cells. However, monomeric species are also seen in the samples that were pooled for subsequent cryo-EM grid\npreparations. I understand that image processing was started with an initial set of particles from which the software was\ntrained to define those particles as templates containing only dimers, before picking the complete set of particles across all\nmicrographs. Do the authors also see monomer particles in their cryo-EM preparations and micrographs? Would it be\npossible to process these particles like dimer particles, keeping in mind the size of these particles is perhaps too small for a\nsingle particle cryo-EM approach?\nd) In absence of the nanobody 1 (Nb1), the resolution of the maps was very limited, whereas in the presence of Nb1, the\noverall resolution of the four maps is high (between 2.6 and 3 angstroms). Thus, the presence of Nb1 certainly stabilizes the\ncomplexes. Although Nb1 facilitated particle classification and alignment, its density was not resolved in the final maps. The\nauthors suggest that Nb1 binds to a relatively flexible part of V1aR. Based on the stability of the complexes, is this\nconclusion not paradoxical?\ne) To my opinion, the comparison of the different antagonist-bound V1aR structures is very interesting and is not sufficiently\ndiscussed. In supplementary figure 6, the structure of V1aR bound by atosiban, balovaptan and SRX246 were\nsuperimposed, but only the receptor is shown. Once the three structures are aligned based on the receptor structure, would it\nbe possible to overlap the three ligands? I think it would be very useful to zoom on this ligand overlap and to present this\nillustration as a main figure. Comparing the docking poses of the three ligands in the orthosteric binding pocket in a single\npanel would be highly informative and would strengthen the discussion part on receptor antagonism and subtype selectivity.\nSince the orthosteric binding pocket is well conserved in the vasopressin/oxytocin receptor family, the binding sites of\natosiban, balovaptan and SRX246 could be compared to that of retosiban in OTR, and those of tolvaptan and\nmambaquaretin in V2R.", "reviewer_2": "This paper presents the first high-resolution cryo-EM structures of the human V1aR in its apo state and in complex with three\nclinically relevant antagonists, atosiban, balovaptan, and SRX246. A major finding is the dimeric assembly of V1aR, which\ncontrasts with previously resolved monomeric structures of related receptors and most of other class A GPCRs, providing\ncrucial insights validated by cell-based assays. The study also elucidates significant conformational changes in ECL2, which\ntransitions from a \"flat\" conformation in the apo state to an \"up\" conformation upon antagonist binding, creating a\nligand-accommodating cavity. Furthermore, the structures and associated functional assays detail the distinct binding modes\nof the antagonists and identify key molecular determinants governing V1aR antagonism and subtype selectivity. This\ncomprehensive mechanistic framework for V1aR regulation and assembly offers invaluable insights, paving the way for\nstructure-guided design of novel antagonists.\nHowever, while the structural data are of high quality and the reported V1aR dimer interface is intriguing, several major\nconcerns regarding receptor function and pharmacology studies significantly limit the mechanistic impact and physiological\nrelevance of the findings.\n1. The only assay used to characterize the engineered V1aR as V1aRcryo is the 3H-AVP radioligand binding assay. This\ndoes not assess whether the receptor is functionally competent. It is critical to perform signaling assays such as Ca2+\nmobilization or G protein coupling to demonstrate that the engineered receptor retains signaling capacity. Without such data,\nit is unclear whether the receptor represents a physiologically relevant signaling-competent conformation.\n2. For the radioligand binding assay, the saturation binding curve for 3H-AVP binding to V1aRcryo (Supplementary Fig. 2a)\ndoes not reach a plateau and appears nearly linear across the concentration range tested. This calls into question the\nreliability of the reported Kd values and suggests that AVP binding is severely compromised (not slightly compromised).\nAccurate Kd determination under equilibrium conditions is essential for subsequent pharmacological analyses. It is\ncommended to include more data points in the 3H-AVP saturation binding assay.\n3. The competition binding assays for the antagonists are also highly problematic. The authors show that all three\nantagonists exhibit similar IC50s (higher IC50 for balovaptan) for V1aRcryo compared to wtV1aR, and state that “Notably,\nthe binding of antagonists atosiban, balovaptan, and SRX246 to V1aRcryo, which are the focus of the current study, were\nalmost unaltered” (line 109). However, according to basic pharmacological principles, if AVP binding is compromised (i.e.,\nincreased Kd), and the IC50 values for antagonists are unchanged, it implies that the Kd values for the antagonists must also\nbe compromised to the same extent. The authors should calculate and report the Kd values of all three antagonists using the\nCheng-Prusoff equation.\n4. This issue also applies to the mutagenesis studies. In the mutagenesis studies, comparisons of IC50 values across V1aR\nmutants are presented without confirming whether AVP binding is preserved in each mutant. This is a critical oversight, as\nchanges in AVP affinity would influence competition binding results. The proper approach is to first determine AVP Kd for\neach mutant and then calculate the corresponding antagonist Kd values to allow meaningful interpretation.\n5. The observed dimer interface is structurally interesting, but its physiological significance remains uncertain. Class A\nGPCR dimerization is still a controversial topic. It has been well established that class A GPCRs do form clusters or loose\noligomers under physiological conditions. However, whether they form stable dimers and how the oligomerization of class A\nGPCRs affect their signaling is still elusive. The authors should consider whether the observed dimerization may result from\ncryo-EM sample preparation artifacts such as the nanobody or the receptor modifications. In this regard, functional\nvalidation, such as ligand binding or signaling assays for dimer interface mutants like K52A and A59Y, is essential to\ndemonstrate the relevance of dimerization to receptor function.\nMoreover, it is important to report whether monomeric particles or highly oligomerized particles were also observed in the\ncryo-EM analysis. Please reference the recent study reporting higher-order oligomeric structures of a class A GPCR\n(doi.org/10.1038/s41594-024-01397-1). This would help clarify whether V1aR forms obligatory dimers, as seen in class C\nGPCRs, or if dimerization represents just one of several possible oligomeric states.\n6. Previous studies have reported dimeric or oligomeric structures of other class A GPCRs, including the\napelin receptor (Nat Struct Mol Biol 2022; Nat Commun 2024) and CXCR4 (Nat Commun 2023; Cell Rep 2025). Comparing\nthe V1aR dimer interface with these receptors would help contextualize the findings and clarify whether this dimerization mode\nis unique or shared.\nOther minor issues\n7. It would be highly informative to discuss ligand selectivity in more details, as it is an important issue for developing\nantagonists for the AVP and OT receptor family. Atosiban is commonly used as a OXTR antagonist.\n8. Since structures of V2R-AVP and OXTR-OT are available, it would be informative to model the binding of AVP and OT to\nV1aR and discuss its preference for the vasopressin and oxytocin peptides.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "In this manuscript, Zhong et al. present the structures of human V1aR in its apo state and in complexes with different\nantagonists. Overall, the manuscript is well-written. I am not a structural biologist and so am only commenting on the single\nmolecule photobleaching experiment in Figure 2 b-f which I believe needs additional clarification and replicates to\ndemonstrate statistical robustness.\nBriefly - the authors perform single molecule photobleaching experiments in fixed cells that are transiently expressing GFP-\ntagged versions of the WT and K52A/A59Y versions of their protein. They conclude that the WT protein shows\npredominantly two-step photobleaching whereas the mutant protein shows single step photobleaching. Below are\ncomments and suggestions for improvement:\nMajor Comments:\n1) The authors describe a need to maintain “very-low” expression levels to minimize overexpression artifacts, yet it does not\nappear that they actually quantify the expression levels in cells. How do the fluorescently tagged protein levels compare to\nendogeneous levels in WT cells?\n2) It would be helpful if the authors could confirm that the GFP-tagged constructs are still functionally active. E.G. do cells\nexpressing the GFP-tagged constructs respond similarly to agonism/antagonism compared to the non-fluorescently tagged\nproteins?\n3) The authors show a representative image in Figure 2c, but do not state what the cell in the image is expressing.\nFurthermore, they indicate that only spots within the yellow circles were chosen for further analysis, but they do not state how\nthese spots were selected or why they were chosen instead of the many other puncta-like objects in their image. I’m also\nconcerned that the representative image shows many large aggregates of protein in addition to single diffraction-limited\nspots. What are these large aggregate structures? Are they an oligomerization artifact due to the tagged protein?\n4) There are no error bars on Figure 2 e or f, nor do the authors provide information about the sample size (number of spots,\nnumber of cells, nor number biological replicates). Are these plots from just a single ROI in a single cell each?\n5) At least in my experience, it is very unusual to have single molecule photobleaching defined by only monomers or dimers\nand not have any higher-order oligomerization states (even if only at a small percentage). Were these curves discarded from\nthe analysis?\n6) There is no information in the methods about how the number of bleaching steps for each trajectory was determined. Was\nthis just done manually from the traces or did the authors utilize some objective criteria or algorithm?"}, "author_response": "Point-by-point response to reviewers’ comments\nWe sincerely appreciate the time and expertise dedicated by all the reviewers whose constructive\ncomments have significantly strengthened our work. In response, we have carefully addressed all\npoints raised using additional experiments and have revised the manuscript to incorporate the\nsuggestions. All changes have been incorporated and are highlighted in the revised submission.\nBelow is a point-by-point response to the reviewers (our response is in blue):\nReviewer #1 (Remarks to the Author):\n[...]\nReply: Thank you for your suggestion. For the apo-state, we collected more images (from 4860\nimages to 8642 images now), which increases the final particles from 319,985 to 504,370. The\noverall resolution of the cryo-EM map has been improved from 2.9 Å to 2.8 Å. While the\nimprovement in global resolution is modest, the local map quality has been significantly\nenhanced.\nUsing the new map of the apo state, the majority of ECL2 was reliably modeled, supported by\nwell-defined local densities (please refer to Fig. 3b for local densities and Supplementary Fig.\n6c for the exact protein sequence included in the final model). The model of the apo-state\nactually includes the protein segments of ECL2 that form the antiparallel beta-sheet observed in\nthe antagonist-bound states (Supplementary Fig. 6c). Interestingly, in the apo structure, these\nregions adopt alpha-helical conformations: one becomes a continuation of TM4, while the other\ninserts into the ligand-binding pocket (Fig. 3a, b, Supplementary Fig. 6c). The two cysteines\n(one in TM3, and the other in ECL2) that form a disulfide bond in the antagonist bound states are\nboth clearly resolved in the cryo-EM density in the apo form (Fig. 3b). They are spatially\nseparated, with bulky residues such as F207 and W111 positioned between them (Fig. 3b). This\narrangement provides strong structural evidence that the two cysteines remain unpaired in the\napo state.\nThe region of ECL2 missing in the apo-state structure (residues 193-201; improved compared to\nthe previous version) is not part of the antiparallel beta-sheet, but the flexible loop connecting the\ntwo beta-sheet segments. This region in the apo-state is visible as continuous and complete\ndensities in the 6Å low-pass-filtered map (Supplementary Fig. 5f; the map has been uploaded\nfor your evaluation). However, due to the limited resolution in this region, reliable atomic\nmodeling is difficult, and thus, this segment was not included in the final model. Nevertheless,\nthe absence of this region in the final model does not affect our conclusion that ECL2 in the apo-\nstate adopts a planar orientation relative to the membrane in the apo structure, and thus is\ndesignated as the “flat” conformation.\nIndeed, the majority of disulfide bonds are formed in the ER. However, the oxidative\nextracellular environment, along with cell surface protein disulfide isomerase, can also facilitate\ndisulfide bond formation or rearrangement, as demonstrated in studies (PMID: 22653978). In the\ncase of GPCRs, a recent study on GPR4 (PMID: 39753131) demonstrated that ECL2 in the apo\nstate has unpaired cysteines. We speculate that this apo state is a ground state. The transition\nfrom alpha-helical to beta-sheet could be very dynamic, which allows the selective recognition of\nthe right ligand, while minimizing nonspecific interactions, particularly because the ligand-\nbinding cavity in the “up” conformation is exceptionally large and exposed.\nWe modified Fig. 3b, c, made new Supplementary Fig. 6c/Supplementary Fig. 5f, and\nmodified text (lines 265-276, lines 282-284) to accommodate the new changes.\n2/ minor criticisms:\na) If we look at the density map of the apo state, there is a density on the intracellular side at the\ndimer interface between TM1 and TM7 which has not been assigned. This density is present in\nthe two copies of the V1aR, symmetrically. Would this density correspond to a phospholipid? To\na cholesterol molecule? Could this density correspond to part of helix 8 which is actually not\nseen?\nReply: Thank you for your question. We also noticed this density on the intracellular side. Based\non the shape and size, we think it might be cholesterol (Please see the figure below for fitting).\nTo be more conservative on the modeling building, we keep it unassigned.\nb) Again, in the apo state structure, if we overlap the density and the corresponding three-\ndimensional model at the level of the extracellular loop 3 (ECL3), it is quite difficult to fit the\nresidues in the density. I think some residues of the ECL3 should be removed.\nReply: Thank you for your suggestion. We reexamined the ECL3 density in the new map of the\napo state and removed residue 325 from the final model.\nc) All the structures solved in this study are those of dimeric V1aR. Indeed, size-exclusion\nchromatography (supplementary figure 1) and SDS-PAGE of the cryo-EM samples show\npurified dimeric species, in agreement with the homodimers seen in live cells. However,\nmonomeric species are also seen in the samples that were pooled for subsequent cryo-EM grid\npreparations. I understand that image processing was started with an initial set of particles from\nwhich the software was trained to define those particles as templates containing only dimers,\nbefore picking the complete set of particles across all micrographs. Do the authors also see\nmonomer particles in their cryo-EM preparations and micrographs? Would it be possible to\nprocess these particles like dimer particles, keeping in mind the size of these particles is perhaps\ntoo small for a single particle cryo-EM approach?\nReply: Thank you for your question. We also observed a small population of particles with a\nsmaller diameter in raw images, likely corresponding to the monomeric species. We have tried to\nuse different templates as well as particle diameter settings to select as many particles as possible\nduring particle picking. However, due to their small size and low abundance, these particles were\nvery likely lost during subsequent data processing and do not generate a reliable 3D\nreconstruction.\nWe modified the text (lines 192-195) to accommodate the new changes.\nd) In absence of the nanobody 1 (Nb1), the resolution of the maps was very limited, whereas in\nthe presence of Nb1, the overall resolution of the four maps is high (between 2.6 and 3\nangstroms). Thus, the presence of Nb1 certainly stabilizes the complexes. Although Nb1\nfacilitated particle classification and alignment, its density was not resolved in the final maps.\nThe authors suggest that Nb1 binds to a relatively flexible part of V1aR. Based on the stability of\nthe complexes, is this conclusion not paradoxical?\nReply: Thank you for your question, and we apologize for any confusion. The limited resolution\nin the absence of Nb1 primarily arises from the lack of a sufficient fiducial mask outside the\ndetergent micelle to guide particle alignment, which is a very common issue in cryo-EM studies\nof apo GPCRs. Although Nb1 is not well resolved in the final high-resolution reconstructions,\nthe 6Å low–pass–filtered map reveals extra densities adjacent to the cytosolic face of V1aR that\nclosely match the shape of Nb1 (Supplementary Fig. 5e). This indicates that Nb1 binds to a\nconformationally heterogeneous epitope on the intracellular side of the receptor. This binding\nposition is also far away from the dimer interface, making it unlikely that Nb1 stabilizes the\ncomplexes.\nWe therefore infer that Nb1 here primarily serves as a fiducial mask, facilitating early particle\nclassifications and global low-resolution alignment. However, its flexible orientation relative to\nthe receptor leads to partial signal averaging and eventual loss of nanobody density during high-\nresolution refinement.\nWe made a new Supplementary Fig. 5e and modified the text (lines 169-176) to explain this\nbetter.\ne) To my opinion, the comparison of the different antagonist-bound V1aR structures is very\ninteresting and is not sufficiently discussed. In supplementary figure 6, the structure of V1aR\nbound by atosiban, balovaptan and SRX246 were superimposed, but only the receptor is shown.\nOnce the three structures are aligned based on the receptor structure, would it be possible to\noverlap the three ligands? I think it would be very useful to zoom on this ligand overlap and to\npresent this illustration as a main figure. Comparing the docking poses of the three ligands in the\northosteric binding pocket in a single panel would be highly informative and would strengthen\nthe discussion part on receptor antagonism and subtype selectivity. Since the orthosteric binding\npocket is well conserved in the vasopressin/oxytocin receptor family, the binding sites of\natosiban, balovaptan and SRX246 could be compared to that of retosiban in OTR, and those of\ntolvaptan and mambaquaretin in V2R.\nReply: Thank you very much for your suggestion. We have now introduced a new main figure,\nFig. 6, to compare the binding poses of different antagonists, which include the three antagonists\nin the current study as well as retosiban (OTR), tolvaptan (V2R), and mambaquaretin (V2R). We\nalso discussed in detail the specific interactions of these antagonists with the receptors, illustrated\nin the newly made Supplementary Fig. 13. The corresponding text has been added as a\ndedicated section (lines 466-488) in the Result section, as well as in the Discussion section\n(Lines 568-586).\nReviewer #2 (Remarks to the Author):\n[...]\nReply: Thank you very much for your comment. The engineering of V1aR into V1aRcryo was\nadopted from a previous structural study of OTR (PMID: 32832646). In the previous study, eight\nmutations/truncations as well as fusion proteins were required to obtain the crystals, and the\nstructure provides very valuable insights into the mechanism of OTR antagonism by retosiban. In\nour study, we aimed to preserve the wild-type sequence as closely as possible and significantly\nreduced the equivalent mutations from 8 to 2. The two adopted mutations used previously in\nOTR, S341C and N344K, were found to be essential for obtaining a stable sample suitable for\nstructural studies; without them, V1aR remained refractory to high-resolution structure\ndetermination.\nIn the previous report (Logan, Richard Thomas (2013). The structure and function of the\nvasopressin V1a receptor. University of Birmingham. Ph.D. Table 4.3), the N344K mutation of\nV1aR was found not to be able to induce downstream signaling. We also used an NFAT-driven\nluciferase assay to assess the G-protein signaling of V1aRcryo and found it to be signaling-\nincompetent (Please see figure below).\nHowever, V1aRcryo retains high sensitivity to antagonists atosiban, balovaptan, and SRX246\n(Please see “reply to comment 3” below), validating its structural integrity. Since the current\nstudy is focused on the molecular mechanism of antagonism, we deemed this engineering\nnecessary and reasonable. To better inform the readers of the properties of V1aRcryo, we\nupdated the text in (Lines 125-140).\n2. For the radioligand binding assay, the saturation binding curve for 3H-AVP binding to\nV1aRcryo (Supplementary Fig. 2a) does not reach a plateau and appears nearly linear across the\nconcentration range tested. This calls into question the reliability of the reported Kd values and\nsuggests that AVP binding is severely compromised (not slightly compromised). Accurate Kd\ndetermination under equilibrium conditions is essential for subsequent pharmacological analyses.\nIt is commended to include more data points in the 3H-AVP saturation binding assay.\nReply: Thank you very much for your suggestion. To improve the accuracy of the Kd\nmeasurement of AVP for V1aRcryo, we included more data points using higher concentrations of\n3H-AVP (Supplementary Fig. 2a). The updated binding curve now reaches a plateau, and the\nnew Kd value is ~19nM, compared with ~13nM in the previous version of the manuscript. The\nKd value for V1aRcryo is updated in Table 3. In light of this change, we updated the description\nof AVP binding from “slightly compromised” to “modestly compromised” to better reflect the\nmagnitude of the change and provide readers with a better understanding of the property of\nV1aRcryo.\nWe updated Supplementary Fig. 2a, Table 3, and modified the text (line 136) to accommodate\nthe new changes.\n3. The competition binding assays for the antagonists are also highly problematic. The authors\nshow that all three antagonists exhibit similar IC50s (higher IC50 for balovaptan) for V1aRcryo\ncompared to wtV1aR, and state that “Notably, the binding of antagonists atosiban, balovaptan,\nand SRX246 to V1aRcryo, which are the focus of the current study, were almost unaltered” (line\n109). However, according to basic pharmacological principles, if AVP binding is compromised\n(i.e., increased Kd), and the IC50 values for antagonists are unchanged, it implies that the Kd\nvalues for the antagonists must also be compromised to the same extent. The authors should\ncalculate and report the Kd values of all three antagonists using the Cheng-Prusoff equation.\nReply: Thank you for your comments, and we apologize for this issue. Based on the updated Kd\nvalue for AVP, we converted the IC50 values for antagonists into Ki using the Cheng-Prusoff\nequation. These values are now reported in the revised Supplementary Table 3. In light of this\nchange, we updated the statement of antagonists binding from “almost unaltered” to “V1aRcryo\nremains highly sensitive to antagonists atosiban, balovaptan, and SRX246, enabling investigation\nof their molecular mechanisms, which are the focus of the current study.”.\nWe updated Supplementary Table 3, and modified the text (lines 138-140) to accommodate\nthe new changes.\n4. This issue also applies to the mutagenesis studies. In the mutagenesis studies, comparisons of\nIC50 values across V1aR mutants are presented without confirming whether AVP binding is\npreserved in each mutant. This is a critical oversight, as changes in AVP affinity would influence\ncompetition binding results. The proper approach is to first determine AVP Kd for each mutant\nand then calculate the corresponding antagonist Kd values to allow meaningful interpretation.\nReply: Thank you for your comments, and we apologize for this oversight. To address this\nconcern, we determined the Kd value of AVP for each mutant using the ligand-binding assays\n(Supplementary Fig. 9, Table 3) and confirmed that the AVP binding for each mutant is\npreserved, with no substantial changes compared to wild-type V1aR. Then, we converted the\nprevious IC50 values into Ki using the Cheng-Prusoff equation (Supplementary Table 3). All\nprevious interpretations of the data remain the same.\nWe made a new Supplementary Fig. 9 and modified the Supplementary Table 3 to\naccommodate the changes.\n5. The observed dimer interface is structurally interesting, but its physiological significance\nremains uncertain. Class A GPCR dimerization is still a controversial topic. It has been well\nestablished that class A GPCRs do form clusters or loose oligomers under physiological\nconditions. However, whether they form stable dimers and how the oligomerization of class A\nGPCRs affect their signaling is still elusive. The authors should consider whether the observed\ndimerization may result from cryo-EM sample preparation artifacts such as the nanobody or the\nreceptor modifications. In this regard, functional validation, such as ligand binding or signaling\nassays for dimer interface mutants like K52A and A59Y, is essential to demonstrate the relevance\nof dimerization to receptor function.\nMoreover, it is important to report whether monomeric particles or highly oligomerized particles\nwere also observed in the cryo-EM analysis. Please reference the recent study reporting higher-\norder oligomeric structures of a class A GPCR (doi.org/10.1038/s41594-024-01397-1). This\nwould help clarify whether V1aR forms obligatory dimers, as seen in class C GPCRs, or if\ndimerization represents just one of several possible oligomeric states.\nReply: Thank you very much for the suggestion. We are not the first to report the existence of\nV1aR dimers in the AVP/OT family. There has been a substantial amount of evidence showing\nthe dimer formation among the AVP/OT family (PMID: 12554793) and their roles in beta-\narrestin association and trafficking (PMID: 14757828), and this has guided the design of\nsymmetric antagonists (PMID: 27420737). All of these underscore the physiological significance\nof the dimer and its importance for pharmacology. Our current study provides the structural\ninsights into the dimer formation.\nFollowing the reviewer’s suggestion, we used the NFAT-driven luciferase assay to assess the G-\nprotein signaling of wild-type V1aR and the K52A/A59Y mutant. The EC50 for AVP is 5-fold\nhigher for the K52A/A59Y mutant than for wild-type, and the IC50 values for all tested\nantagonists are higher for the mutant as well (Supplementary Figure 7, Table 4), which\nsuggests that the dimer is physiologically relevant.\nWe did observe a small population of particles with a smaller diameter in raw images, likely\ncorresponding to the monomeric species. However, due to their small size and low abundance,\nthese particles were very likely lost during subsequent data processing and do not generate a\nreliable 3D reconstruction. In contrast, no clear trimeric or tetrameric particles were detected in\nour cryo-EM datasets, unlike CXCR4, for which recent cryo-EM studies have visualized trimeric\nand tetrameric assemblies. Now, we reported those findings in the text and referenced the paper\nof CXCR4 as well.\nWe made a new Supplementary Fig. 7, Table 4, and modified the text (lines 192-197, lines 247-\n249) to accommodate the new changes.\n6. Previous studies have reported dimeric or oligomeric structures of other class A GPCRs,\nincluding the apelin receptor (Nat Struct Mol Biol 2022; Nat Commun 2024) and CXCR4 (Nat\nCommun 2023; Cell Rep 2025). Comparing the V1aR dimer interface with these receptors would\nhelp contextualize the findings and clarify whether this dimerization mode is unique or shared.\nReply: Thank you very much for your suggestion. We now compared the dimer interface of\nV1aR with that of β1AR, rhodopsin, CXCR4, GPR3, and APJR and illustrated the comparison in\nthe newly made Supplementary Fig. 8 and contextualized our findings. The corresponding text\nhas been added in the Result section (lines 250-256).\nOther minor issues\n7. It would be highly informative to discuss ligand selectivity in more details, as it is an\nimportant issue for developing antagonists for the AVP and OT receptor family. Atosiban is\ncommonly used as a OXTR antagonist.\nReply: Thank you very much for your suggestion. We have now discussed the selectivity in more\ndetail in the Discussion section (lines 568-586) and made a new Supplementary Fig. 13 to make\nit more informative. Atosiban’s dual antagonism is noted and discussed.\n8. Since structures of V2R-AVP and OXTR-OT are available, it would be informative to model\nthe binding of AVP and OT to V1aR and discuss its preference for the vasopressin and oxytocin\npeptides.\nReply: Thank you for your suggestion. We generated OT-bound V1aR and AVP-bound V1aR\nusing Modeller based on V2R-AVP and OTR-OT structures (Supplementary Fig. 11). Using the\ncomputationally generated models, we discussed the residues that might contribute to the\npreference of AVP over OT. Phe³ of AVP inserts more deeply into the TM5–TM6 hydrophobic\ncleft than Ile³ of oxytocin, enhancing hydrophobic interactions with V1aR. Moreover, Met2005.43\nof V1aR will clash with Ile³(oxytocin), whereas in OTR and V2R, isoleucine at this position can\naccommodate Ile³(oxytocin).\nWe made a new Supplementary Fig. 11 and modified the text (lines 385-396) to accommodate\nthe new changes.\nReviewer #3 (Remarks to the Author):\n[...]\nReply: We sincerely thank Reviewer #3 for the thoughtful evaluation of our manuscript. As an\nEarly Career Researcher participating in Nature Communications’ peer review training initiative,\nyour perspective is invaluable to the scientific community. We appreciate the rigor of your\nmethodological insights. Your contributions exemplify how emerging scholars enhance academic\ndiscourse, and we commend the journal’s commitment to fostering new voices in peer review.\nReviewer #4 (Remarks to the Author):\n[...]\nReply: Thank you for your comment. V1aR exhibits very low, or even negligible, expression in\nthe common cell lines available to us. We acknowledge that we did not perform absolute\nquantification to directly compare protein levels in our experimental system with those in a\nsubset of cells from normal tissues, as this is technically very challenging. Conventionally,\nGPCR dimerization is assessed using BRET assays following 24-48 hours of transfection, an\napproach that has also been applied to confirm the dimer formation of AVP/OT family members\n(PMID: 12554793). However, higher expression levels increase the likelihood of dimer or even\nhigher-order oligomer formation. Therefore, we attempted to probe the dimerization behavior of\nV1aR under a much lower expression level compared with the traditional method. In our\nexperimental setup of the single-molecule photobleaching approach, the transfection period was\nrestricted to 3 hours, a substantially shorter duration, under which V1aR remained predominantly\nin the dimeric state.\n2) It would be helpful if the authors could confirm that the GFP-tagged constructs are still\nfunctionally active. E.G. do cells expressing the GFP-tagged constructs respond similarly to\nagonism/antagonism compared to the non-fluorescently tagged proteins?\nReply: Thank you for your suggestion. GFP-tag was changed into mStayGold-tag for better\nphotostability behaviors. We used the NFAT-driven luciferase assay to assess the G-protein\nsignaling of wild-type V1aR and the mStayGold-tagged version. In all cases, the mStayGold-\ntagged construct responds similarly to agonism by AVP and antagonism by\natosiban/balovaptan/SRX246, compared to the wild-type proteins (Supplementary Fig. 7).\nWe made a new Supplementary Fig. 7, Table 4, and modified the text (lines 207-209) to\naccommodate the new changes.\n3) The authors show a representative image in Figure 2c, but do not state what the cell in the\nimage is expressing. Furthermore, they indicate that only spots within the yellow circles were\nchosen for further analysis, but they do not state how these spots were selected or why they were\nchosen instead of the many other puncta-like objects in their image. I’m also concerned that the\nrepresentative image shows many large aggregates of protein in addition to single diffraction-\nlimited spots. What are these large aggregate structures? Are they an oligomerization artifact due\nto the tagged protein?\nReply: Thank you for these insightful questions, and we apologize for the missing information.\nIn the updated Fig. 2c, the cell is expressing mStayGold-tagged V1aR K52A/A59Y (previously\nshown as mEGFP-tagged WT V1aR in the original figure). This information is now provided in\nthe legend of the updated Fig. 2c.\nUsing a single-molecule photobleaching assay to distinguish monomers from dimers proved to\nbe extremely challenging for two main reasons:\n(1) Stringent requirement for low expression levels. The expression level needs to be\nsufficiently low to minimize both oligomerization and overlap of individual molecules.\nWe anticipated that stable expression systems or even CRISPR knock-in would lead to\nlarge aggregates, which would preclude reliable photobleaching analysis. Therefore, as\nmentioned above, we restricted the transient transfection period to less than 3 hours.\nDuring imaging, we manually inspected the fluorescence intensity of individual cells and\nselected those with relatively low fluorescence signals to reduce the probability of\noverlapping molecules. However, even under these conditions, expression levels still\nvaried between cells within the same sample. Moreover, within a single cell, protein\ndensity was not uniform across the entire plasma membrane. Consequently, even in low-\nexpression cells, many fluorescent spots likely correspond to large protein aggregates,\nwhich is a common caveat of transient transfection; these spots were excluded from\nfurther analysis.\n(2) Photobleaching during cell screening reduces the apparent dimer fraction. As\ndescribed above, we needed to manually inspect cells to identify those with suitably low\nexpression levels, which were relatively rare. During this inspection, photobleaching\ninevitably occurred. This process preferentially reduces the number of detectable dimeric\nevents: once one fluorophore in a dimer bleaches, the remaining signal becomes\nindistinguishable from that of a monomer. In contrast, the number of monomeric events is\nlargely unaffected, because spots that are fully photobleached are simply not selected for\nanalysis. This concern motivated our switch from mEGFP to mStayGold, the most\nphotostable fluorescent protein available to date. Although the photostability of\nmStayGold is significantly compromised after fixation, this change allowed us to obtain\nmore data of sufficient quality for statistical analysis. Nevertheless, the fraction of\ndimeric events observed in our study is still likely underestimated.\nOverall, the puncta-like objects that were excluded from analysis most likely represent protein\naggregates caused by transient transfection. However, they are not artifacts of the tagging using\nmStayGold, as we have confirmed that the mStayGold-tagged constructs retain normal function.\nFurthermore, we have additional evidence supporting that WT V1aR predominantly exists in a\ndimeric state as observed in the cryo-EM structures. The dimeric assembly was functionally\nvalidated using an NFAT-driven luciferase assay to assess the G-protein signaling of wild-type\nV1aR and the K52A/A59Y mutant. The EC50 for AVP is 5-fold higher for the K52A/A59Y\nmutant than for wild-type, and the IC50 values for all tested antagonists are higher for the mutant\nas well (Supplementary Fig. 7, Table 4), which suggests that the observed dimer is\nphysiologically relevant.\n4) There are no error bars on Figure 2 e or f, nor do the authors provide information about the\nsample size (number of spots, number of cells, nor number biological replicates). Are these plots\nfrom just a single ROI in a single cell each?\nReply: Thank you for the great question. After switching from mEGFP to mStayGold, we were\nable to obtain more data of sufficient quality for statistical analysis (300-400 events in total). In\nthe updated Fig. 2e and 2f, the data are presented as mean ± SEM. For each group, the data were\nobtained from three cells across two independent experiments. This information has been\nincluded in the legend of Fig. 2 and in the Supplementary Information.\n5) At least in my experience, it is very unusual to have single molecule photobleaching defined\nby only monomers or dimers and not have any higher-order oligomerization states (even if only\nat a small percentage). Were these curves discarded from the analysis?\nReply: Thank you for the great question. We did observe a small fraction of spots that exhibited\nmore than two photobleaching steps in their fluorescence intensity profiles (less than 10% of all\ninspected spots). However, as mentioned above, transient transfection inevitably led to non-\nuniform protein expression and overlapping molecules. Consequently, we could not reliably\ndetermine whether spots with more than two steps represented genuine higher-order oligomeric\nevents, overlapping molecules, or simply protein aggregates. Because there is currently no\nevidence that WT V1aR forms higher-order oligomers beyond dimers, we chose to exclude all\nspots that exhibited more than two photobleaching steps from our analysis.\nWe have now explicitly mentioned this point in the Supplementary Information.\n“Disputed spots, including those showing more than two photobleaching steps or lacking clear,\nwell-defined transitions in their intensity profiles, were excluded from further analysis.”\n6) There is no information in the methods about how the number of bleaching steps for each\ntrajectory was determined. Was this just done manually from the traces or did the authors utilize\nsome objective criteria or algorithm?\nReply: Thank you for your comment. Because we were unable to identify a reliable algorithm\nfor automatic analysis, we manually inspected the fluorescence intensity profiles to determine\nthe number of photobleaching steps. We have now added a detailed description of how the\nnumber of bleaching steps was determined in the Supplementary Information under “Single-\nmolecule photobleaching assay”.\n“Raw image stacks comprising 3,000 frames were acquired at a frame rate of 5 Hz. Two\nresearchers independently selected approximately 100 non-overlapping, single-spot regions from\neach stack using ImageJ software. The summed fluorescence intensity within a 3×3-pixel region\nof interest (ROI), centered on each spot, was computed. Fluorescence intensity profiles across\nthe entire stack were then plotted to identify photobleaching steps for individual spots. One\nresearcher manually classified the bleaching curve types, and a second researcher performed an\nindependent review. Disputed spots, including those showing more than two photobleaching\nsteps or lacking clear, well-defined transitions in their intensity profiles, were excluded from\nfurther analysis. The number of spots exhibiting distinct bleaching steps was quantified for each\nexperimental group and subjected to statistical analysis. Each group included three replicates\nderived from two independent experiments.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised version of the manuscript by Zhong P et al. describes the cryo-electron microscopy (cryo-EM) three-dimensional (3D) structure of the apo state of the human vasopressin V1a receptor (V1aR) and those of the receptor in complex with three different types of antagonists. These are the first high-resolution structures of V1aR ever described. Interestingly, all structures presented in this manuscript are those of homodimeric V1aR particles. In addition, the authors show that the apo state structure of V1aR displays a flat extracellular loop 2 (ECL2) including an alpha helix, a significant difference in conformation with that of the antagonist-bound receptor states (in which this ECL2 exhibits a typical antiparallel beta-sheet). This study provides a comprehensive understanding of V1aR assembly and opens the way to structure-guided development of new antagonists for treating central nervous system disorders. It also gives information about antagonist receptor selectivity.\nAll the criticisms and comments I raised previously were adequately addressed in the revised version. Also, the remarks from the other reviewers were addressed (to my personal point of view).\nMy main criticism was focused on the ligand free state of the V1aR, for which the density map of the ECL2 was missing. Due to this absence, the previous results were not in agreement with the conclusions. Thanks to the authors who collected more cryo-EM images and describe now a conformation of the ECL2 which supports their conclusions. Indeed, the number of particles was increased, the overall resolution of the cryo-EM map was improved, and more importantly the majority of ECL2 was reliably modeled. This improvement allowed the authors to show that ECL2 adopts an alpha-helical conformation in the apo state and inserts into the ligand-binding pocket. These new data are now discussed, particularly about the transition from alpha-helical (apo state) to beta-sheet (ligand-bound) conformations upon ligand binding. New figures or new panels were also added to accomodate these changes.\nAmong the minor comments, since the orthosteric binding pocket is well conserved in the vasopressin/oxytocin receptor family, I also asked for a comparison between the binding sites of atosiban, balovaptan and SRX246 in the V1aR with those of retosiban in the OTR and those of tolvaptan and mambaquaretin in the V2R. The authors now introduced a new main figure and a new Supplementary figure, as well as text in the Result and Discussion sections.\nAll other minor criticisms were addressed adequately. And consequently, text and/or illustrations were added to the revised version.\nI still have one remark about reference 39. Either it is incomplete, or it is wrong. Please complete or correct.\nProviding this last modification (reference 39), I consider the manuscript is now acceptable for publication in Nature Communications.", "reviewer_2": "The authors have addressed most of my concerns with additional pharmacological assays, and the new data look fine. I have only one remaining minor concern. The authors note that the two introduced mutations disrupt the signaling capacity of V1aR, although ligand binding is largely unaffected. In many GPCR structural studies that used engineered receptors with impaired signaling, the structural insights typically focused on ligand binding. In this case, the discussion of the potential dimer interface should be more cautious. It would be helpful to include a brief discussion of this limitation in the manuscript.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have generally addressed my comments by more clearly detailing the methods used for their selection of which cells and photobleaching spots to quantify, by more clearly describing their quantification procedure, and by verifying that the mStayGold-tagged proteins are functional. I do find their logic in describing the origin of and for excluding the quantification of large aggregates to be a bit circular.\nFor example, the authors claim in the rebuttal that \"stable expression systems or even CRISPR knock-in would lead to large aggregates, which would preclude reliable photobleaching analysis\". Yet, they also state that \"Overall, the puncta-like objects that were excluded from analysis most likely represent protein aggregates caused by transient transfection.\" So, if I understand the argument correctly, the authors anticipate that both endogenous expression (at native protein levels) and transient over expression lead to aggregates?\nHowever, they exclude any large spots or traces with more than 2 photobleaching steps from further analysis, \"Because there is currently no evidence that WT V1aR forms higher-order oligomers beyond dimers\". I find this argument very confusing.\nHowever, the authors do clearly state in the revised methods that they select only spots that show dimer or monomer photobleaching curves and discard any traces with more than 2 photobleaching steps. Given that this is a relatively minor part of the manuscript, I feel that this is probably sufficient."}, "author_response": "Reply: Thank you very much for you time and expertise in improving this manuscript. Reference\n39 is a dissertation. We have manually made it more complete.\nReply: Thank you very much for you time and expertise in improving this manuscript. We now\nadded a sentence in the disscussion section to discuss the potential limitation. (whether the\ndimeric assembly persists within the signaling complex remains to be seen.)\nReply: Thank you very much for you time and expertise in improving this manuscript.\nReply: We sincerely thank you for your time and expertise in helping to improve this\nmanuscript, and we apologize for any confusion caused.\nAs mentioned, there is no available cell line that expresses V1aR at endogeneous level, we had to\nrely on recombinant expression systems, including transient tranfection, stable expression or\nCRIPSR knock-in of an expression cassette. However, these methods typically lead to expression\nlevels much higher than endogenous, often resulting in aggregation of the expressed membrane\nproteins. Notably, there is currently no evidence that wild-type V1aR forms higher-order\noligomers beyond dimers, consistent with our structural data and previous studies. Therefore, a\nfraction of large fluorescent spots is likely attributable to protein aggregates in our over\nexpression system.\nMoreover, the intensity pattern of a single molecule spans an area of approximately 5 × 5 pixels,\ncorresponding to ~600 × 600 nm in this study. Therefore, under our transient overexpression\nconditions, there is still a probability that two to three dimers could fall within the same\ndiffraction-limited region. This explains why a fraction of spots exhibit more than two\nphotobleaching steps.\nWe therefore conclude that regions showing more than two steps are more likely due to higher\nlocal protein density/expression as well as protein aggregates. Accordingly, we excluded large\nspots and any traces displaying more than two photobleaching steps from further analysis."}]

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