Molecular basis of ligand recognition and activation of human V2 vasopressin receptor

Dear Editor,

Vasopressin type 2 receptor (V2R) belongs to the vasopressin (VP)/oxytocin (OT) receptor subfamily of G protein-coupled receptors (GPCRs), which comprises at least four closely related receptor subtypes: V1aR, V1bR, V2R, and OTR.¹ These receptors are activated by arginine vasopressin (AVP) and OT, two endogenous nine-amino acid neurohypophysial hormones, which are thought to mediate a biologically conserved role in social behavior and sexual reproduction.² V2R is mainly expressed in the renal collecting duct principal cells and mediates the antidiuretic action of AVP by accelerating water reabsorption, thereby playing a vital role in controlling water homeostasis. Moreover, numerous gain-of-function and loss-of-function mutations of V2R have been identified and are closely associated with human diseases, including nephrogenic syndrome of inappropriate diuresis (NSIAD) and X-linked congenital nephrogenic diabetes insipidus (NDI).³ Thus, V2R has attracted intense interest as a drug target. However, due to a lack of structural information, how AVP recognizes and activates V2R remains elusive, which hampers the V2R-targeted drug design. Here, we determined a 2.6 Å resolution cryo-EM structure of the full-length, G_s-coupled human V2R bound to AVP (Fig. 1a; Supplementary information, Table S1). The G_s protein was engineered based on mini-G_s that was used in the crystal structure determination of the G_s-coupled adenosine A_2A receptor (A_2AR) to stabilize the V2R–G_s protein complex (Supplementary information, Data S1).⁴ The final structure of the AVP–V2R–G_s complex contains all residues of AVP (residues 1–9), the Gα_s Ras-like domain, Gβγ subunits, Nb35, scFv16, and the V2R residues from T31 to L339^8.57 (superscripts refer to Ballesteros–Weinstein numbering⁵). The majority of amino acid side chains, including AVP, transmembrane domain (TMD), all flexible intracellular loops (ICLs) and extracellular loops (ECLs) except for ICL3 and G185–G188 in ECL2, were well resolved in the model, refined against the EM density map (Fig. 1a; Supplementary information, Figs. S1–S3). The complex structure can provide detailed information on the binding interface between AVP and helix bundle of the receptor, as well as the receptor–G_s interface.

Fig. 1: Cryo-EM structure of the AVP–V2R–G_s complex.

a Orthogonal view of the density map (left panel) and model (right panel) for the AVP–V2R–G_s complex. V2R, slate blue; AVP, green; G_s, orange; Gβ, salmon; Gγ, cyan; Nb35, gray; scFv16, magenta; cholesterols, yellow. b, c Interactions between AVP and residues in V2R-binding pocket. d, e A comparison of the conformational change of ECL1 and ECL2 in AVP-bound V2R structure and retosiban-bound OTR (PDB: 6TPK) inactive structure. f Conformational changes of TM6 and conserved W^6.48 and Y/F^6.44 of V2R compared to inactive OTR. Red arrows indicate the movement of TM6 and the side chains of W^6.48 and Y/F^6.44 in V2R relative to inactive OTR. g Conformational change of TM7 in AVP–V2R structure relative to inactive OTR, β₂AR–G_s, and A_2AR–G_s structures. Red arrow indicates the movement of TM7 in V2R. h Sequence comparison of conserved P^5.50-I^3.40-F^6.44 motif among V2R, V1aR, V1bR, OTR, A_2AR, and β₂AR. Polar residues are colored in red and nonpolar residues in green. Conformational changes of AVP-bound V2R structure resulted from substitutions in the conserved P^5.50-I^3.40-F^6.44 motif relative to inactive OTR (i) and active β₂AR (j) structures, respectively. k, l A conformational comparison of receptor helical bundle and Gα_s α5 helix among AVP–V2R–G_s, β₂AR–G_s, and A_2AR–G_s complexes. Red arrows indicate the movements of intracellular tips of TMs5, 6, and α5 helix of Gα_s from V2R–G_s relative to other complexes. Black arrows label the cytoplasmic ends of TM5 in these structures. H-bonds and salt bridges are depicted as red and green dashed lines, respectively. The disulfide bonds are shown as yellow sticks. The structural alignment is based on receptors.

AVP occupies an orthosteric binding pocket in the TMD bundle composed of all TM helices and ECLs (Fig. 1b, c; Supplementary information, Table S2). The most notable conformational feature of AVP is the tocin ring formed by a disulfide bridge between the first and sixth cysteine residues, presenting a “spoon-like” conformation (Supplementary information, Fig. S4a, b). The cyclic spoon head inserts deeply into the TMD core, while the C-terminal spoon tail stretches toward the ECLs of the receptor. Cys^1P of AVP and Q96^2.61, K116^3.29, Q119^3.32 of V2R form a stabilizing H-bond network, consistent with the decreased potency of the receptor mutants with these residues mutated (Fig. 1b; Supplementary information, Fig. S4c, d). The hydroxyl group of Tyr^2P forms an H-bonds with the main chain oxygen of L312^7.40, whereas its main chain CO group forms another H-bond with Q174^4.60 (Fig. 1b; Supplementary information, Fig. S4c). Phe^3P buries in a hydrophobic cleft constituted by M120^3.33, M123^3.36, Y205^5.38, V206^5.39, I209^5.42, F287^6.51, F288^6.52, and Q291^6.55 (Fig. 1b; Supplementary information, Fig. S4c). Other polar contacts are seen between Gln^4P and R202^5.35, Gln^4P and the backbone and side chain oxygens of Q291^6.55, as well as Asn^5P and the main chain of A194^ECL2 (Fig. 1b, c; Supplementary information, Fig. S4c). Arg^8P forms salt bridges with D33^1.28 and E40^1.35, the latter forms an extra salt bridge with K100^2.65 (Fig. 1c; Supplementary information, Fig. S4c). Additionally, the C-terminal amide of AVP points to ECL1, stacking against the R104^ECL1 guanidinium group and H-bonding to the backbone CO group of D103^ECL1 (Fig. 1c; Supplementary information, Fig. S4c). These interactions between AVP and residues in V2R binding pocket were confirmed through alanine mutagenesis studies using cAMP accumulation assays (Supplementary information, Fig. S4d–g and Table S4).

This structure also provides a template to study the selectivity of AVP and OT. OT has two amino acid substitutions (Phe^3P to Ile^3P and Arg^8P to Leu^8P) (Supplementary information, Fig. S5a), and exhibits a much lower selectivity against V2R as opposed to AVP.⁶ By docking OT into the V2R binding pocket, a weaker hydrophobic interaction network is created for Ile^3P compared to its equivalent residue Phe^3P in AVP (Supplementary information, Fig. S5b). Additionally, the hydrophobic leucine at position 8 no longer shows the polar interactions with the receptor as compared with Arg^8P of AVP (Supplementary information, Fig. S5c). Thus, the peptide-binding pocket of V2R defines a relatively unfavorable binding environment for OT in contrast to AVP.

A “DCWA” sequence in ECL2 is highly conserved throughout VP/OT receptor subfamily but is not a feature of other class A GPCRs.⁷ Interestingly, structural comparison of the AVP–V2R–G_s complex with inactive OTR (PDB: 6TPK)⁸ reveals a notable conformational transition of ECL2 from β-hairpin to a flexible loop (Fig. 1d). In this conserved sequence, W193^ECL2 and A194^ECL2 directly contact with AVP. Asn^5P may push and stack against W193^ECL2 and overcome the probable steric clash between Asn^5P and W188^ECL2 of OTR, the cognate residue of V2R, which may confer this conformational transition (Fig. 1d). Although D191^ECL2 and C192^ECL2 do not directly interact with AVP, the former forms an H-bond to the F105^ECL1 amide and a salt-bridge to R104^ECL1 and the latter forms a conserved disulfide bond with C112^3.25. These interactions, in turn, stabilize ECL1 and ECL2 conformations, thereby facilitating the interactions between ECL1 and the C-terminal amide of AVP (Fig. 1e; Supplementary information, Fig. S4f, g and Table S4).

A structural comparison of the AVP–V2R–G_s complex with the inactive OTR supports the contention that our structure is indeed in the active state. AVP triggers the rotameric change of “toggle switch” W284^6.48, initiating the rotation and a notable outward displacement of TM6, the hallmark of GPCR activation (Fig. 1f; Supplementary information, Fig. S6a).⁹ Intriguingly, a distortion of TM7 helix was observed between L312^7.40 and S315^7.43. This distorted conformation appears to be stabilized by the H-bond between Tyr^2P of AVP and the main chain CO group of L312^7.40 in the receptor, drawing TM7 toward the peptide-binding pocket core (Fig. 1g). [Phe2]AVP, a synthesized AVP analog with a substitution of Tyr by Phe that abolishes this H-bond, showed decreased agonistic activity by 28-fold,¹⁰ implying that the polar interaction between Tyr^2P and the receptor is critical to AVP-induced V2R activation. Specifically, F^6.44 and I^3.40, residues in the conserved “P-I-F” motif in class A GPCRs, are substituted by polar residues Y280^6.44 and S127^3.40 in V2R, respectively (Fig. 1h). AVP binding triggers the rotation of Y280^6.44 relative to the inactive OTR (Fig. 1i), subsequently forming a featured H-bond network between Y280^6.44/S127^3.40 and the backbone CO group of V213^5.46 in contrast to active β₂AR (PDB: 3SN6),¹¹ which probably stabilizes the active conformation of the receptor (Fig. 1j). The disease-associated mutations S127^3.40F and Y280^6.44C in V2R deactivate the receptor,^12,13 while the replacement of F284^6.44 in OTR by the V2R-equivalent tyrosine converts AVP from a partial to a full agonist,¹⁴ supporting the hypothesis that these unconventional “P-I-F” residues play critical roles in V2R activation. Furthermore, the cytoplasmic end of TM4 is one helical turn shorter than that of the inactive OTR,⁸ G_s-coupled β₂AR¹¹ and A_2AR⁴ (PDB: 5G53), thereby releasing a longer ICL2. The ICL2 protrudes towards TM1 and adopts a C-shaped conformation (Supplementary information, Fig. S6b). This C-shaped loop is stabilized by H-bonds formed between H80^2.45 and the backbone CO group of H155^ICL2, as well as W71^ICL1 and backbone CO group of A154^ICL2, and further by an intra-loop polar interaction network (Supplementary information, Fig. S6c–e), which may maintain ICL2 in a specific conformation and probably affect the ICL2–Gα_s interaction pattern.

TM5 and TM6 with the outward movement of the cytoplasmic ends open a cytoplasmic cavity together with TM2, TM3, and helix 8 to accommodate the α5 helix of Gα_s, constituting a major V2R–G_s interface (Supplementary information, Fig. S7a). One additional interface relates to ICL2 that interacts with α5 helix, αN-β1 junction, and the top of the β3-strand of Gα_s, presumably stabilized by hydrophobic contacts (Supplementary information, Fig. S7b). In contrast to the G_s-coupled β₂AR and A_2AR structures, the cytoplasmic ends of TM5 and TM6 in V2R display a smaller amplitude of outward displacements (5.8 Å for TM5 and 8.7 Å for TM6 relative to β₂AR,¹¹ 2.5 Å for TM5 and 7.2 Å for TM6 relative to A_2AR,⁴ measured at Cα carbon of residues 5.67 and 6.30, respectively) (Fig. 1k, l). Also, a noticeable shift of G_s protein in the AVP–V2R–G_s complex may partly be caused by relatively inward positions of TM5 and TM6 that push the entire G_s heterotrimer shifts in the same direction. Besides, the α5 helix of Gα_s in the AVP–V2R–G_s complex shifts half a helical turn away from the 7TM core (Fig. 1k). These structural differences altogether create a smaller cytoplasmic cavity to accommodate G_s protein. Indeed, the solvent-accessible surface area (SASA) of the V2R–Gα_s interface (943.32 Ų) is smaller than that of β₂AR–Gα_s (1,030 Ų) and A_2AR–Gα_s interfaces (1,276 Ų) (Supplementary information, Fig. S7c). Interestingly, compared to G_s-coupled β₂AR and A_2AR, V2R exhibits a 1–2 helical turns shorter TM5 cytoplasmic end, which may be caused by a consecutive P-G-P sequence (P238, G239, and P240, residues known as α-helix breakers), resulting in fewer interactions between TM5 of V2R and Gα_s subunit (Fig. 1l; Supplementary information, Fig. S8).

In conclusion, we solved the cryo-EM structure of G_s-coupled V2R bound to its endogenous ligand AVP. In this complex, AVP presents a unique cyclic conformation formed by an intramolecular disulfide bond and engages the orthosteric binding pocket of V2R in a ligand-specific mode. AVP-induced distortion of TM7 and a unique polar network formed by equivalent residues in the “P-I-F” motif may differentiate activation of V2R from other class A GPCRs. A smaller amplitude of the outward displacement of TM5 and TM6 and the concomitant shift of G_s protein not only distinguish V2R from its counterparts but also represent a diversified G_s coupling mechanism. Additionally, our structure is valuable to the mechanistic understanding of V2R mutation-associated diseases (Supplementary information, Fig. S9), while systematic investigations are required to enrich our knowledge in this regard. Together, our findings provide a framework for understanding AVP recognition and V2R activation, thereby offering a structural template for drug design targeting V2R.

Accession codes

The corresponding coordinates and cryo-EM density map have been deposited in the Protein Data Bank (http://www.rcsb.org/pdb) with code 7DW9, and in EMDB (http://www.ebi.ac.uk/pdbe/emdb/) with code EMD-30877.