Structural basis of antagonist selectivity in endothelin receptors

Hou, Junyi; Liu, Shenhui; Zhang, Xiaodan; Tu, Guowei; Wu, Lijie; Zhang, Yijie; Yang, Hao; Li, Xiangcheng; Liu, Junlin; Jiang, Longquan; Tan, Qiwen; Bai, Fang; Liu, Zhijie; Miao, Changhong; Hua, Tian; Luo, Zhe

doi:10.1038/s41421-024-00705-9

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Published: 30 July 2024

Structural basis of antagonist selectivity in endothelin receptors

Junyi Hou¹^na1,
Shenhui Liu^2,3^na1,
Xiaodan Zhang^2,3^na1,
Guowei Tu¹^na1,
Lijie Wu²,
Yijie Zhang¹,
Hao Yang^3,4,
Xiangcheng Li^3,4,
Junlin Liu²,
Longquan Jiang^2,3,
Qiwen Tan²,
Fang Bai^3,4,
Zhijie Liu ORCID: orcid.org/0000-0001-7279-2893^2,3,
Changhong Miao^5,6,
Tian Hua ORCID: orcid.org/0000-0002-6132-2068^2,3 &
…
Zhe Luo^1,7,8

Cell Discovery volume 10, Article number: 79 (2024) Cite this article

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Abstract

Endothelins and their receptors, ET_A and ET_B, play vital roles in maintaining vascular homeostasis. Therapeutically targeting endothelin receptors, particularly through ET_A antagonists, has shown efficacy in treating pulmonary arterial hypertension (PAH) and other cardiovascular- and renal-related diseases. Here we present cryo-electron microscopy structures of ET_A in complex with two PAH drugs, macitentan and ambrisentan, along with zibotentan, a selective ET_A antagonist, respectively. Notably, a specialized anti-ET_A antibody facilitated the structural elucidation. These structures, together with the active-state structures of ET-1-bound ET_A and ET_B, and the agonist BQ3020-bound ET_B, in complex with G_q, unveil the molecular basis of agonist/antagonist binding modes in endothelin receptors. Key residues that confer antagonist selectivity to endothelin receptors were identified along with the activation mechanism of ET_A. Furthermore, our results suggest that ECL2 in ET_A can serve as an epitope for antibody-mediated receptor antagonism. Collectively, these insights establish a robust theoretical framework for the rational design of small-molecule drugs and antibodies with selective activity against endothelin receptors.

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Introduction

Endothelins are pivotal regulators of cardiovascular functions, essential for maintaining vascular tone and overall cardiovascular homeostasis¹. Three endothelin peptides, namely ET-1, ET-2, and ET-3, are characterized by two unique cysteine–cysteine crosslinks² and activate the endothelin receptor (ETR) subtypes ET_A and ET_B^3,4. Notably, human ET_A and ET_B receptors share 63% sequence homology³ but differ significantly in their ligand affinity and function. For instance, ET_A preferentially binds ET-1 and ET-2 over ET-3⁵, mediating strong vasoconstriction, whereas ET_B exhibits equal affinity for all three isoforms, primarily inducing vasorelaxation through nitric oxide and facilitating ET-1 clearance^6,7. Consequently, ETRs are crucial targets for the treatment of cardiovascular diseases⁸.

Given the complex role of ETRs, the development of therapeutic drugs has primarily focused on vasodilatory antagonists for treating pulmonary arterial hypertension (PAH) and autoimmune diseases^9,10. Notable selective ET_A antagonists or dual ET_A/ET_B antagonists include bosentan, macitentan, and ambrisentan^{11,12,13,14,15}. Additionally, ETR antagonists like sparsentan, aprocitentan, and zibotentan are currently under investigation for their potential efficacy in treating refractory hypertension and various kidney diseases^16,17,18,19. Conversely, ET_B agonists are being explored for therapeutic benefits such as vasodilation and neuroprotection^20,21. Furthermore, the development of therapeutic vaccines and monoclonal antibodies targeting ET_A represents an exciting frontier in PAH treatment, combining high specificity with a reduced risk of side effects²². Preclinical studies have shown promise in these approaches to decrease pulmonary arterial pressure^23,24.

Structural studies on ETRs have elucidated the mechanisms of endothelin ligand recognition and activation. Extensive X-ray crystallography work has revealed the interaction patterns within the ET_B receptor when bound with various ligands, including ET-1, ET-3, and several antagonists^{5,25,26,27,28,29}. Complementarily, recent cryo-electron microscopy (cryo-EM) studies on ET_A and ET_B in complex with G protein in active states have provided insights into the conserved recognition mechanisms for endogenous agonists and the selectivity for synthetic agonists between ET_A and ET_B³⁰. Despite these advances, the structural basis for ET_A antagonism remains less understood, a gap that limits the design of selective antagonists. In addition to small-molecule antagonists, a monoclonal antibody (Fab₃₀₁) specifically targeting ET_A is currently in phase Ib clinical trials²³. The specificity of antibodies may address the selectivity issues associated with small-molecule antagonists for ETRs^22,24. However, there is currently no structural information on antibody–receptor complexes^24,31.

This study aims to address these questions by presenting cryo-EM structures of human ET_A in complex with key small-molecule antagonists and Fab₃₀₁ and by detailing the activation mechanisms of both ET_A and ET_B receptors. These molecular insights are crucial for advancing the development of therapeutics for conditions such as PAH and are part of broader efforts to enhance the specificity and efficacy of ETR-targeted drugs.

Results

Structure determination of antagonist-bound ET_A structures

To understand the structure basis for the antagonist binding modes in ET_A, we analyzed three distinct compounds — macitentan, ambrisentan and zibotentan — measuring their activities on ET_A in calcium mobilization assay (Fig. 1a; Supplementary Fig. S1). Determining the structure of inactive-state G protein-coupled receptors (GPCRs) via cryo-EM is inherently challenging due to the absence of the heterotrimeric G protein, which is essential for particle alignment in active GPCR structure determination. Here, we developed an optimized strategy to solve the inactive ET_A structures. Initially, to overcome the low surface expression of ET_A, endoglucanase H was fused to the N-terminus of ET_A, along with N- and C-terminal truncations (Materials and methods). Then, to compensate for the absence of the G protein, a thermostabilized apocytochrome b562 RIL (BRIL)³² protein was fused between TM5 and TM6, replacing ET_A’s third intracellular loop (ICL3) (Supplementary Fig. S1a). The design of the BRIL fusion sites for uninterrupted helicity was informed by AlphaFold2³³ predictions.

**Fig. 1: Cryo-EM structures of ET_A in complex with macitentan, ambrisentan, zibotentan.**

Subsequent stabilization of the modified ET_A construct involved the introduction of a BRIL-binding Fab and a nanobody (Nb) that reinforces the hinge region, as previously described^34,35. Despite these modifications, we encountered stability and orientation preferences during cryo-EM grid preparation. To address these issues, we introduced an ET_A-specific antibody, Fab₃₀₁²³, which not only conferred additional stability to the complex but also exhibited antagonistic effects against ET-1-induced ET_A signaling. These strategic modifications enabled us to determine the cryo-EM structures of Fab₃₀₁-bound ET_A in complex with macitentan, ambrisentan and zibotentan, at nominal resolutions of 3.1 Å, 3.2 Å and 3.2 Å, respectively (Fig. 1b−e; Supplementary Fig. S2 and Table S1). The high-resolution cryo-EM maps provided detailed electron density in the orthosteric ligand-binding pocket, enabling accurate placement of each antagonist (Supplementary Fig. S2). It is noteworthy that the interaction interface between Fab₃₀₁ and ET_A is quite limited, and within the cryo-EM particles, Fab₃₀₁ appears to exhibit a degree of movement.

Comparative analysis of the macitentan–ET_A, ambrisentan–ET_A and zibotentan–ET_A complexes reveals a conserved conformation among the three structures, with C_α root mean square deviation (RMSD) values between 0.7 Å and 1.0 Å (Supplementary Fig. S3a, b). Compared to the crystal structures of antagonist-bound ET_B, both receptors adopt a similar inactive conformation, with an RMSD of 1.5 Å (Supplementary Fig. S3c).

Binding of macitentan to ET_A

Macitentan, designed as an ET_A-selective antagonist, is a derivative of the dual-acting antagonist bosentan. In our calcium mobilization assay, macitentan shows a high affinity to ET_A, with an IC₅₀ of 1.3 nM (Fig. 1a; Supplementary Table S2), contrasting to the lower affinity to ET_B at 14.5 μM (Supplementary Fig. S4b), which is also consistent with the previous research results³⁶. Structurally, macitentan retains the pyrimidine core characteristic of bosentan but distinguishes itself with a sulfonamide substitution at the fourth position³⁷ (Supplementary Fig. S1b, c). The binding of macitentan within the ET_A pocket involves several distinct interactions (Fig. 2a). The sulfonamide moiety in macitentan establishes a hydrogen bond with R326^6.55 (Fig. 2b), and forms ionic interactions with residues K166^3.33, K255^5.38 and R326^6.55 from ET_A (Fig. 2b). The bromophenyl group is embedded in the receptor’s hydrophobic core, forming hydrophobic interactions with residues W319^6.48, V169^3.36, Y263^5.46, H323^6.52, and L259^5.42 from TMs 3, 5, and 6 (Fig. 2c), and is further stabilized by a cation–π interaction with K166^3.33 (Fig. 2b). The 2-(5-bromopyrimidin-2-yl)oxyethoxy component, linked to the sixth position of the pyrimidine, allows the oxygen atom in the bromopyrimidine to form a hydrogen bond with Q165^3.32 (Fig. 2d). Importantly, this moiety inserts deeper into the orthosteric pocket than the similar groups in bosentan or K-8794 in ET_B, positioned to form a halogen bond with D126^2.50 (Fig. 2d). Furthermore, bromopyrimidine engages in π–π interactions with Y129^2.53 and the “toggle switch” residue W319^6.48 (Fig. 2d). The ethyl tail attached to the sulfonamide moiety is nestled within a hydrophobic pocket formed by F161^3.28, P162^3.29, and F224^4.64 from TM3 and TM4 of ET_A. Prior research has demonstrated that substituting the sulfonamide moiety with sulfamide significantly boosts ETR antagonists’ receptor affinity¹¹. Our structural analysis reveals that the addition of the -NH- group in macitentan contributes to a more stable electrostatic network with K166^3.33 and K255^5.38, enhancing macitentan’s binding affinity (Fig. 2b). Notably, the R326^6.55A mutation showed minimal influence on macitentan’s activity on ET_A, aligning with macitentan’s stronger electrostatic network at this location in ET_A (Fig. 2m).

**Fig. 2: Characterization of macitentan, ambrisentan, and zibotentan binding modes in ET_A.**

Binding of ambrisentan to ET_A

Ambrisentan, a propionic acid derivative discovered through high-throughput screening, differentiates itself from macitentan by featuring a carboxylic acid group instead of a sulfonamide moiety¹⁵ (Supplementary Fig. S1d). Despite its smaller molecular weight relative to macitentan and zibotentan, ambrisentan exhibits a remarkably high affinity to ET_A, befitting its role as an ET_A-selective antagonist (Fig. 1a). The structural elucidation of its binding to ET_A uncovers essential interactions. The carboxylic acid group forms ionic interactions with the positively charged residues K166^3.33 and R326^6.55 (Fig. 2f), while a network of hydrogen bonds with residues R326^6.55, K166^3.33, and Q165^3.32 firmly anchors ambrisentan in the binding site (Fig. 2e, f). Moreover, the hydrophobic interaction of one benzene ring from the symmetric pair in ambrisentan with residues F161^3.28, P162^3.29, and F224^4.64 creates a snug fit within a subpocket (Fig. 2f). The counterpart benzene ring engages with residues L322^6.51, W319^6.48, I355^7.39, and A358^7.42 spanning TM6 and TM7 (Fig. 2g). Additionally, the methyl groups on the pyrimidine ring of ambrisentan adapt to the hydrophobic cores formed by residues V169^3.36, L259^5.42, Y263^5.46, and H323^6.52 from TMs 3, 5 and 6 (Fig. 2h), further stabilizing the antagonist binding within the receptor. Furthermore, our molecular dynamics (MD) simulations indicate the stable binding of ambrisentan within the pocket (Supplementary Fig. S5).

Binding of zibotentan to ET_A

Zibotentan, a sulfonamide-based compound, exhibits selective antagonism towards ET_A with an IC₅₀ of 8.6 nM in our calcium mobilization assay (Fig. 1a). Structurally distinguished by a central pyridine ring flanked by two bulky substituents (Fig. 2i), zibotentan adopts a chair-like conformation within the orthosteric binding pocket, contrasting with the spatial arrangement of macitentan (Supplementary Fig. S1c, e). The sulfonamide group forms electrostatic interactions with Q165^3.32, K166^3.33 and R326^6.55 (Fig. 2j), whereas the pyrazinyl rings, modified with methoxy and methyl groups, engage in hydrophobic contacts with residues around W319^6.48 from TMs 3, 5 6 (Fig. 2k). On the opposite end, the 1,3,4-oxadiazol-2-yl segment of zibotentan extends towards ECL2, forming a π–π interaction with F161^3.28 and a hydrogen bond with K140^2.64 (Fig. 2l). Furthermore, Y129^2.53 appears to participate in polar interactions or hydrogen bonds with the pyridine ring’s nitrogen (Fig. 2l).

Subsequent structural investigations suggest that zibotentan’s selectivity for ET_A may be significantly influenced by the residue F161^3.28, which is a valine (V177^3.28) in ET_B. This phenylalanine F161^3.28 acts as a “tray” that stabilizes one end of the zibotentan molecule (Fig. 2l). Concordantly, mutation of F161^3.28A or F161^3.28V significantly reduces zibotentan’s activity on ET_A (Fig. 2n). Additionally, the interaction with Y129^2.53 is also crucial; its alteration to phenylalanine or histidine (Y129^2.53F or Y129^2.53H) markedly reduces zibotentan’s activity on ET_A, highlighting the importance of this residue for antagonist specificity (Supplementary Fig. S4c−e). Intriguingly, introducing corresponding mutations into ET_B (V177^3.28F and H150^2.53Y) partially impairs the activity of zibotentan on ET_B, which underscores the critical role of these residues in the determination of antagonist selectivity in ET_A or ET_B subtype (Fig. 2o).

Structure basis of antagonist selectivity in ET_A and ET_B

Structural elucidation of ET_A in complex with various antagonists, each featuring distinct scaffold architectures, advances our understanding of the antagonist binding modes in ET_A and delineates commonalities critical for their antagonistic function. An important aspect of their activity is the interaction with the positively charged region in ET_A’s orthosteric pocket. Functionally important groups, such as the sulfonylamide in bosentan, macitentan, and zibotentan, along with the carboxylic acid group in ambrisentan, are essential. These functional groups occupy the position corresponding to W21, the C-terminal end of the endogenous agonist ET-1 (Fig. 3a), a key engagement site with ET_A³⁰. Another shared trait of these antagonists is their interaction in close proximity to TM5 and TM6, which helps stabilize ET_A conformation in this region (Fig. 3b). The three antagonist-bound structures display a hydrophobic moiety in this vicinity, forming hydrophobic contacts with residues from TMs 3, 5 and 6 (Fig. 3b). These groups also hinder the inward movement of the side chain of W319^6.48 through hydrophobic or π–π interactions (Fig. 3b). Mutagenesis data, such as L259^5.42A, Y263^5.46A or L322^6.51A, further confirm the critical role of these hydrophobic contacts in sustaining antagonist efficacy (Supplementary Fig. S4f−h).

Regarding the selectivity differences, we observed that, in ET_A, F161^3.28 rotates inwards to the orthosteric pocket in the antagonist-bound state, which results in a compact antagonist-binding pocket in ET_A (Fig. 3c). In contrast, in the corresponding site in ET_B, a smaller V177^3.28 yields a more expansive antagonist-binding pocket (Fig. 3d). This spatial variation accounts for the more effective accommodation of the larger bulky hydrophobic group (4-t-butylphenyl) of bosentan within ET_B, while the smaller hydrophobic groups of macitentan and ambrisentan show a preference for ET_A (Fig. 3e, f). Regarding zibotentan’s ET_A selectivity, as previously discussed, this may be attributed to the different residue Y129^2.53 in ET_A compared to H150^2.53 in ET_B (Figs. 2l, 3g). Together, Y129^2.53 and F161^3.28 in ET_A may partially account for the observed selectivity of antagonists toward ET_B.

Active and inactive conformation features of ETRs

To delve deeper into the activation mechanisms of ETRs, we determined the structures of ET_A and ET_B in their active states, at global resolutions of 3.3 Å for ET-1-bound ET_A–miniG_s/q–Nb35 complex and 3.2 Å for ET_B–miniG_s/q–Nb35 complex (Fig. 4a, b; Supplementary Fig. S6 and Table S1). In addition, a structure of ET_B complexed with the selective agonist BQ3020³⁸, was determined at a resolution of 3.0 Å (Fig. 4c; Supplementary Fig. S6a and Table S1). The overall conformations of ET-1-bound ET_A–miniG_s/q and ET_B–miniG_s/q complex structures, including the ET-1 binding poses, are consistent with previously reported ETR structures³⁰ (Supplementary Fig. S7a, b). Notably, the binding pose of BQ3020 in ET_B closely resembles that of ET-1, with a C_α RMSD of 0.8 Å for the receptor (Fig. 4d, f). BQ3020, which differs from the ET_B-selective agonist IRL1620 by a single residue (Fig. 4e), exhibits an overall structural similarity, as the BQ3020–ET_B and IRL1620–ET_B complex structures display an RMSD of 0.9 Å. This indicates the aligned positioning of the agonists within the binding pocket (Fig. 4d). In the BQ3020–ET_B complex, BQ3020’s C-terminal configuration closely resembles that of ET-1, albeit with a slightly downward shift in the α-helix within the binding pocket, due to the absence of disulfide-bond constraints (Fig. 4d).

**Fig. 4: Cryo-EM structures of BQ3020–ET_B–miniG_s/q–Nb35, ET1–ET_B–miniG_s/q–Nb35, and ET1–ET_A–miniG_s/q–Nb35 complexes.**

Additionally, the mutation W146^ECL1A in ET_A does not significantly affect receptor activation, whereas the analogous mutation W167^ECL1A in ET_B results in a substantial reduction in ET_B activation (Supplementary Fig. S7c and Table S3). This suggests that ET_B requires interactions with larger hydrophobic groups at this site for activation. Within ET_B’s ECL1, F169^ECL1 is posited to engage in a π–π interaction with W167^ECL1 (Supplementary Fig. S7d), a key conformation for receptor function. Alanine mutation of F169^ECL1 hampers ET-1’s capability to activate ET_B (Supplementary Fig. S7c).

Employing the Residue–Residue Contact Score (RRCS) tool³⁹, we analyzed structures bound by three antagonists, confirming that the residue contacts are characteristics of the inactive-state class A GPCRs (Supplementary Fig. S8). A comparative analysis of the macitentan-bound ET_A structure against the ET-1-bound ET_A structure reveals significant conformational changes when ET_A transits from the inactive to active states. ET-1 binding promotes an inward movement of the extracellular portions of TM2, TM6 and TM7, resulting in a more compact receptor core (Fig. 5a). Concurrently, ECL2 moves inwards substantially, acting as a “lid” that secures ET-1 in place, facilitated by a π–π interaction between ET-1’s Y13 and Y231^ECL2 in ET_A (Fig. 5a). However, we cannot exclude the influence of Fab₃₀₁ on the conformational changes of ECL2 when compared to the state bound alone by the small-molecular antagonist. The EM density map allowed the modeling of extended N-terminal residues, revealing a tighter packing with ECL2 and ECL3 in the ET-1-bound ET_A structure compared to the macitentan-bound state (Fig. 5a). These structural rearrangements lead to a more compact orthosteric pocket for ET-1 interaction. The characteristic outward displacement of the cytoplasmic part of TM6 by 9.7 Å in the ET-1-bound ET_A–miniG_s/q structure, reflects the conformational changes, indicative of class A GPCR activation (Fig. 5b). Moreover, the intracellular portion of TM7 undergoes a displacement of ~2.9 Å (measured by the C_α atom of L369^7.53), in the active state (Fig. 5c).

**Fig. 5: Structural comparison of Fab₃₀₁–macitentan–ET_A–anti-BRIL Fab-Nb and ET1–ET_A–miniG_s/q–Nb35 complexes.**

Mechanism for ET_A activation

The antagonist-bound ET_A structures illuminate the underlying activation mechanism. Upon ET-1 binding, the side chain of I19 residue exerts pressure on I355^7.39, prompting a downward shift of this residue and the associated intracellular half of TM7 (Fig. 5d). Concurrently, ET-1’s W21 residue engages with the “toggle switch” residue W319^6.48 (Fig. 5e). This interaction induces a downward rotation of W319^6.48, facilitating a hydrogen bond formation between its nitrogen atom and N361^7.45 (Fig. 5e). The downward motions of W319^6.48 and N361^7.45 promote an inward-to-outward rotation of F315^6.44 within the P^5.50I/V^3.40F^6.44 motif (Fig. 5e, f), triggering the outward movement of TM6 at the cytoplasmic end (Fig. 5e). These sequential events are accompanied by the disruption of interactions between L176^3.43, L311^6.40 and V312^6.41, which results in the release of the stacking of TM3 and TM6, further promoting the outward swing of TM6 (Fig. 5g). Additionally, the downward movement of N361^7.45 leads to the collapse of the Na⁺ pocket, previously stabilized by D126^2.50, T172^3.39, and N361^7.45 in the inactive state, and triggers subsequent rearrangements between TM7, TM3 and TM2 (Fig. 5h).

Regarding the highly conserved N^7.49P^7.50xxY^7.53 motif in class A GPCRs, ET_A and ET_B feature a variant of N^7.49P^7.50xxL^7.53Y^7.54, wherein Y^7.53 is replaced by L^7.53. During ET_A activation, L179^3.46 does not engage in a polar inter-helix interaction, which permits L369^7.53 to move downward, enhancing its coupling with the C-terminus of the G protein’s α5 helix (Fig. 5c). Concerning the D^3.49R^3.50Y^3.51 motif, ET_A activation disrupts the conserved ion lock between D182^3.49 and R183^3.50. The released R183^3.50 then forms a polar interaction with Y275^5.58 and establishes interactions with the α5 helix of the G protein, anchoring the receptor in its active state (Fig. 5i).

Insights into antibody design to antagonize ET_A

In our structural analysis of the antagonists-bound ET_A, the employed Fab₃₀₁ shows antagonistic effect and facilitates the structure determination. The EM density maps clearly reveal the binding of Fab₃₀₁ to ET_A’s ECL2 region (Fig. 6a). However, due to the dynamic nature of the interaction between Fab₃₀₁ and ET_A’s ECL2, pinpointing their precise binding interface is challenging. We addressed this by using AlphaFold2 multimer^33,40 prediction to model the Fab₃₀₁–ET_A complex interface (Supplementary Fig. S9a). Ultimately the epitope of Fab₃₀₁ was roughly located based on the fitting of the predicted model with the experimental EM densities (Fig. 6c).

**Fig. 6: Binding interface between Fab₃₀₁ and ET_A.**

To pinpoint the exact ECL2 residues involved in binding, we performed an extensive alanine scanning mutagenesis. Our size exclusion chromatography analysis identified that the region spanning residues 230^ECL2–235^ECL2 in ET_A, and in particularly, residues R232^ECL2 and G233^ECL2, are crucial for Fab₃₀₁ binding. Mutations of R232^ECL2A or G233^ECL2A resulted in a near-complete loss of Fab₃₀₁’s binding to ET_A (Fig. 6b; Supplementary Fig. S10). These results complement our predicted binding mode, where R232^ECL2 of ET_A forms an important polar interaction network with Fab₃₀₁ (Fig. 6c). Moreover, the hydrophobic interactions at this interface are crucial for antibody binding, as confirmed by our calcium mobilization assay on Fab₃₀₁ (Supplementary Fig. S9b). Within this model, E230^ECL2 establishes hydrogen bonds with the CDRL1 and CDRL2 regions of Fab₃₀₁, whereas Y231^ECL2 and E234^ECL2, together with R232^ECL2, participate in a complex hydrogen bond network within the CDRH3 region of Fab₃₀₁ (Fig. 6c). Alanine mutations on E230^ECL2, Y231^ECL2, and R232^ECL2 also affected ET-1’s activity on ET_A. Notably, Y231^ECL2 mutation showed the most significant effect in calcium mobilization assay (Fig. 6d). These data underscore the essential role of ECL2 in the recognition of ligands and Fab₃₀₁ by ET_A.

Comparative structural analysis of ET_A bound by ET-1 and Fab₃₀₁ illustrates how ECL2 residues E230^ECL2, Y231^ECL2, and R232^ECL2, which directly interact with ET-1, undergo a conformational change upon Fab₃₀₁ binding. This change fosters a polar interaction network with Fab₃₀₁, effectively inhibiting ET-1 binding to ET_A (Fig. 6f). Additionally, an ECL2 sequence alignment between ET_A and ET_B uncovers the basis for Fab₃₀₁’s selectivity for ET_A over ET_B (Fig. 6e). The distinct sequence variation, specifically at residues E230–Q235 of ET_A, is pronounced between the two receptors. This divergence provides a valuable template for the design of selective antibodies targeting the ETR subtypes.

Discussion

The structural elucidation of antagonist-bound ET_A in this study provides critical insights into the mechanisms underpinning receptor selectivity and activity modulation. The cryo-EM structures of ET_A in complex with macitentan, ambrisentan, and zibotentan underscore the intricate molecular interactions that govern antagonist binding.

The analysis of antagonist-bound ET_A structures reveals conserved features aligning with the inactive state observed in antagonist-bound ET_B structures, suggesting common structural themes in ETR antagonism. Macitentan’s high affinity to ET_A is attributed to several key interactions, including hydrogen bonds and ionic interactions within the orthosteric pocket. The unique sulfonamide moiety enhances these interactions, resulting in a stable electrostatic network crucial for high binding affinity. Similarly, ambrisentan’s high affinity and selectivity are due to its carboxylic acid group forming robust ionic and hydrogen bonds, coupled with hydrophobic interactions that create a snug fit within the binding pocket. Zibotentan’s unique chair-like conformation and its interactions with residues around TM3, TM5, and TM6 further highlight the structural adaptations that facilitate selective binding.

The selectivity of antagonists for ET_A over ET_B is primarily influenced by specific residues within the binding pocket. F161^3.28 in ET_A, which rotates inward in the antagonist-bound state, creates a compact binding pocket that favors smaller hydrophobic groups, whereas the corresponding V177^3.28 in ET_B accommodates larger groups. The residue Y129^2.53 in ET_A, compared to H150^2.53 in ET_B, further contributes to this selectivity by engaging in critical polar interactions.

The antagonistic effect of Fab₃₀₁ and its ability to stabilize the ET_A structure were utilized to gain further insights into antibody–ET_A interactions. The binding of Fab₃₀₁ to the ECL2 region of ET_A involves critical residues, notably R232^ECL2 and G233^ECL2, essential for Fab₃₀₁’s binding affinity. The detailed mapping of the Fab₃₀₁–ET_A interface provides a template for designing selective antibodies that target specific ETR subtypes.

This study enhances our understanding of the structural basis for antagonist selectivity and activation mechanisms of ETRs. The high-resolution structures of antagonist-bound ET_A reveal conserved features essential for selective binding and receptor stabilization. Key residues within the orthosteric pocket play crucial roles in determining antagonist affinity and selectivity, offering valuable insights into the design of selective therapeutic agents.

Materials and methods

Cell lines

Spodoptera frugiperda (Sf9, expression systems) and Trichoplusia ni (High Five, Thermo Fisher Scientific) cells were gown in ESF medium at 27 °C and 120 rpm. CHO-K1 cells were cultured in Ham’s F-12K (Kaighn’s) Medium (Gibco-Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-Thermo Fisher Scientific), and 100 U/mL Penicillin-Streptomycin (Gibco-Thermo Fisher Scientific) in a humidified incubator at 37 °C with 5% CO₂.

ET_A and ET_B construct design

For the complex structures of G protein-coupled ET_A and ET_B, the human ET_A or ET_B genes were cloned into pFastBac1 vector. This vector was modified to include a hemagglutinin (HA) signal peptide, a Flag tag at the N-terminus of the receptor, an HRV3C protease recognition site, and a 10× His tag at the C-terminus. To improve the protein yield of ET_A, we fused an endoglucanase H (PDB: 2CIT) at the N-terminus of ET_A, simultaneously truncating residues 1–49 at the N-terminus and residues 406–427 at the C-terminus. Similarly, for ET_B, we truncated N-terminal residues 1–66 and C-terminal residues 407–442 and fused a sialidase H (PDB: 2VK5) to the N-terminus.

For the antagonist-bound ET_A structure determination, the cytochrome b562RIL (BRIL) protein was inserted into the ICL3, specifically replacing residues 282–298 of ET_A. Additionally, we appended sequences ARRQL and ERARSTL from A_2A adenosine receptor to the N-terminus and C-terminus of BRIL, respectively⁴¹. The truncations for ET_A were consistent with those described above.

ET_A–miniG_s/q–Nb35 and ET_B–miniG_s/q–Nb35 complex formation and purification

The ET_A–miniG_s/q–Nb35 and ET_B–miniG_s/q–Nb35 complexes were assembled in vitro using modified ET_A and ET_B, miniGα_s/q, Gβ₁γ₂ and Nb35. The expression and purification of miniGα_s/q, Gβ₁γ₂ and Nb35 were performed as previously described⁴². ET_A and ET_B were expressed in Sf9 insect cells using the Bac-to-Bac baculovirus expression system (Invitrogen). The cells were cultured at 27 °C and collected 48 h after infection. Subsequently, 2 L cell pellets were sequentially washed and centrifuged with hypotonic buffer (10 mM HEPES, pH 7.4, 20 mM KCl, 10 mM MgCl₂, EDTA-free protease inhibitor cocktail tablets), followed by a high osmotic buffer (10 mM HEPES, pH 7.4, 1 M NaCl. 20 mM KCl, 10 mM MgCl₂, EDTA-free protease inhibitor cocktail tablets). The purified membrane was solubilized with a buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 1% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace), 0.2% (w/v) cholesterol hemisucinate (CHS, Sigma-Aldrich)) for 2 h at 4 °C. The supernatant was separated by ultracentrifugation for 30 min, and then incubated with TALON resin (Takara) overnight at 4 °C. The resin was washed with a 15-column volume (CV) wash buffer I (25 mM HEPES, pH 7.4,100 mM NaCl, 10% (v/v) glycerol, 0.05% (w/v) LMNG, 0.01% (w/v) CHS, 20 mM imidazole) and a 15-CV wash buffer II (25 mM HEPES, pH 7.4,100 mM NaCl, 10% (v/v) glycerol, 0.01% (w/v) LMNG, 0.002% (w/v) CHS, 30 mM imidazole). Then elution was carried out with a 3-CV buffer (25 mM HEPES, pH 7.4, 100 mM NaCl, 10% (v/v) glycerol, 0.01% (w/v) LMNG, 0.002% (w/v) CHS, 300 mM imidazole). After the protein was eluted, the imidazole was removed by PD MiniTrap G-25 column (GE Health Care). ET_A or ET_B, miniGα_s/q, Gβ₁γ₂ and Nb35 were mixed at a molar mass ratio of 1:1.2:1.2:1.5, incubated at 24 °C for 1 h; and then 1 μL apyrase (0.5 U/μL) were added, followed by an additional 1-h incubation at 25 °C. The mixture samples were loaded onto the Superdex 200 10/300 column (GE Healthcare) in an equilibration buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00015% (w/v) CHS, 0.00025% (w/v) GDN, 100 mM TCEP). The peak fractions containing ET_A– or ET_B–miniG_s/q–β₁γ₂–Nb35 complex were collected and concentrated to 2.0–4.0 mg/mL for cryo-EM sample preparation. 10 μM ET-1 or BQ3020 were added to the buffer during the purification process.

Expression and purification of Fab₃₀₁

As previously reported, Getagozumab²³ was used to produce the Fab₃₀₁ fragment. The Fab₃₀₁ fragment was codon-optimized and synthesized by GenScript. The corresponding light and heavy chain genes were then subcloned into the pFastBac Dual vector for expression using the Bac-to-Bac baculovirus expression system. Hi5 insect cells were infected with baculovirus at a density of 2 × 10⁶ cells per mL and cultured at 27 °C. Cells were harvested 72 h post infection by centrifugation at 2000 rpm for 30 min, and the clear supernatant was collected. The pH of this supernatant was adjusted to 7.0 before the supernatant was applied to a 2 mL Ni-NTA resin and incubated at 4 °C for 2 h. The column was subsequently washed with a 15-CV buffer containing 20 mM HEPES, pH7.0, 500 mM NaCl, and 20 mM imidazole to remove nonspecifically bound proteins. The protein of interest was eluted from the column using an elution buffer containing 20 mM HEPES, pH 7.0, 100 mM NaCl, and 400 mM imidazole. The eluted protein fractions were then further purified on a Superdex 200 10/300 column, which was equilibrated in a buffer containing 20 mM HEPES, pH7.0, 100 mM NaCl, and 10% glycerol. Monomeric fractions were pooled, concentrated to 8.3 mg/mL, flash-frozen in liquid nitrogen, and stored at –80 °C for future use.

Fab₃₀₁–ET_A–anti-BRIL Fab-Nb complex formation and purification

The purification of the ET_A receptor was performed analogously to the method described above, with the addition of one of the antagonists — ambrisentan, macitentan, or zibotentan during purification. The anti-Bril Fab was expressed in mammalian cells and purified following the protocol described previously³⁴. The anti-Fab Nb was expressed in E. coli BL21(DE3) strain and purified according to the previously established methods³⁵.

For complex formation, the ET_A, Fab₃₀₁, anti-BRIL Fab, and anti-Fab Nb were mixed at a molar mass ratio of 1:1.2:1.2:1.5. This mixture was incubated at 4 °C for 4 h, and then concentrated and applied to a Superdex 200 10/300 GL column preequilibrated with the buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00015% (w/v) CHS, 0.00025% (w/v) GDN, 100 mM TCEP, and 50 μM of the chosen antagonist — either ambrisentan, macitentan or zibotentan. Fractions containing the peak of interest were concentrated to an approximate concentration of 12 mg/mL for cryo-EM specimen preparation.

Cryo-EM sample preparation and data collection

A total of 3 μL of each complex sample, BQ3020–ET_B–miniG_s/q, ET1–ET_B–miniG_s/q, ET1–ET_A–miniG_s/q, Fab₃₀₁–macitentan–ET_A, Fab₃₀₁–ambrisentan–ET_A or Fab₃₀₁–zibotentan–ET_A, was applied to glow-discharged 300 mesh alloy grids (CryoMatrix Amorphous alloy film R1.2/1.3), and vitrified by Vitrobot Mark IV (Thermo Fisher Scientific). Excess sample was blotted by a filter paper for 3 s with a blot force of 2 before plunge-freezing in liquid ethane with a FEI Vitrobot Mark IV at 100% humidity and 4 °C. The frozen grids were transferred to liquid nitrogen and stored for data acquisition. Cryo-EM data collection was conducted with the Krios G4 cryo-transmission electron microscope (Thermo Fisher Scientific) operating at 300 kV, equipped with the Falcon 4 Direct Electron Detector (Thermo Fisher Scientific). Movies were recorded at a calibrated magnification of 130,000×, yielding a pixel size of 0.96 Å. A total dose of 60 electrons per square angstrom (e^–/Å²) was administered. Automated data collection was facilitated by the EPU software, utilizing a defocus range spanning from –1.0 μm to –2.0 μm.

Cryo-EM data processing

The overall cryo-EM data processing workflows for the BQ3020–ET_B–miniG_s/q, ET1–ET_B–miniG_s/q, ET1–ET_A–miniG_s/q, Fab₃₀₁–macitentan–ET_A, Fab₃₀₁–ambrisentan–ET_A and Fab₃₀₁–zibotentan–ET_A are shown in Supplementary Figs. S2 and S6. Cryo-EM movie stacks were corrected for beam-induced shifts utilizing the dose-weighting approach in Patch Motion Correction⁴³. The contrast transfer function (CTF) parameters were calculated by employing the patch CTF estimation in CryoSPARC⁴⁴.

For the BQ3020–ET_B–miniG_s/q–Nb35 complex, a total of 7685 images were imported in CryoSPARC v.4.0.1. A conventional neural network-based method Topaz⁴⁵ implemented in CryoSPARC, was used for particle picking. 2,662,705 particles were extracted and then subjected to iterative 2D classification and ab initio reconstruction. Subsequently, 199,360 particles were selected for heterogeneous refinement. The best class was selected for homogeneous refinement, non-uniform refinement, and local refinement, generating a high-quality density map at a resolution of 3.0 Å. DeepEMhancer was applied to enhance local density. The processing steps for the ET1–ET_B–miniG_s/q–Nb35 and ET1–ET_A–miniG_s/q–Nb35 complexes, mirrored this approach, with specific details provided in Supplementary Fig. S6.

For Fab₃₀₁–macitentan–ET_A, Fab₃₀₁–ambrisentan–ET_A and Fab₃₀₁–zibotentan–ET_A complexes, the initial data processing steps were consistent with those of the G protein complexes. However, additional post-processing strategies were implemented. The Fab₃₀₁–macitentan–ET_A complex achieved high-quality density after the standard local refinement; thus, no further optimization was performed. In contrast, for the ambrisentan and zibotentan complexes, masks were generated post local refinement to omit the dynamic regions of Fab₃₀₁. Detailed statistics on the number of images and particles at each processing stage are available in Supplementary Fig. S2.

Model building and refinement

For Fab₃₀₁–macitentan–ET_A, Fab₃₀₁–ambrisentan–ET_A and Fab₃₀₁–zibotentan–ET_A complexes, the AlphaFold2-predicted ET_A was used for the receptor modeling. The initial models for BRIL, anti-BRIL Fab and anti-Fab Nb were based on the structures derived from the GPR183 complex structure (PDB: 7TUY)⁴⁶. For the BQ3020–ET_B–miniG_s/q–Nb35, ET1–ET_B–miniG_s/q–Nb35, and ET1–ET_A–miniG_s/q–Nb35 complexes, model building and refinement of the miniG_s/q and Nb35 began with the miniG_s/q structure from the GPR139–miniG_s/q complex structure (PDB: 7VUH)⁴². Model docking into the EM density maps was carried out using Chimera⁴⁷, followed by iterative manual adjustments and rebuilding in Coot⁴⁸. Subsequent refinement was performed using phenix.real_space_refine in PHENIX⁴⁹. Statistical validation of the model was conducted through MolProbity⁵⁰. Structural figures were prepared using ChimeraX⁵¹. The complete refinement statistics are documented in Supplementary Table S1.

Intracellular calcium mobilization assay

CHO-K1 cells were cultured in Ham’s F-12K (Kaighn’s) Medium (Gibco-Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-Thermo Fisher Scientific), and 100 U/mL Penicillin-Streptomycin (Gibco-Thermo Fisher Scientific) in a humidified incubator at 37 °C with 5% CO₂. Cells were seeded in 6-cm dishes overnight; when the density reached 60%–80%, the cells were transferred to F12K Medium supplemented with 10% FBS and transfected with 1.5 μg DNA encoding the ET_A WT or mutants or 3 μg DNA encoding the ET_B WT or mutants using TransIT2020 (Mirus Biosciences). The next day, transfected cells were harvested from the plate using Versene buffer (Gibco-Thermo Fisher Scientific) and seeded into black-sided, clear-bottomed 384-well plates (Agilent) at a density of 1,5000 cells per well. After 20 h, the 1% dFBS medium was removed, and cells were loaded with 20 μL/well of 1× Calcium 6 dye (Molecular Devices) and incubated at 37 °C for 1 h in the dark. 10 μL/well of 3× ET-1 was added and the plates were read using the FLIPR Tetra High Throughput Cellular Screening System (Molecular Devices). To measure antagonist or Fab potency, 10 μL/well of 3× antagonist or Fab was added and incubated with the cells for 30 min at room temperature. The fluorescence intensity was recorded for 2 min after 10 μL/well of 4× ET-1 addition. All data were analyzed using GraphPad Prism 8 and the data are from at least three independent replicate experiments. All plots are shown as means ± SEM. Data were determined using one-way ANOVA followed by Dunnett’s multiple test compared with WT. The top value was normalized to 100% and the bottom value was normalized to 0% for the final presentation. Nonlinear curve fit was performed using a four-parameter logistic equation (log (agonist vs response) or log (inhibitor vs response)).

MD simulation

MD simulations were performed using the GROMACS2021 software, employing the CHARMM36m force field⁵² and incorporating TIP3 water molecules. Parameters for ambrisentan were derived using the CGenFF force field⁵³. To compensate for the missing ICL3 region in the ambrisentan complex structure, a segment from ET_A in the G protein complex was used. The complete structure was prepared with the Protein Preparation Wizard in Maestro (2023-1, Schrödinger), which included the determination of the protonation states of residues at pH 7.4 using PROPKA. The protein was embedded into a lipid bilayer composing 150 POPC molecules using CHARMM-GUI⁵⁴, which also facilitated the addition of 0.15 M sodium and chloride ions to neutralize the system’s charge. Energy minimization and equilibration processes were performed following the default protocol of CHARMM-GUI, using a cutoff distance of 12 Å for nonbonded contacts and the Particle Mesh Ewald (PME) method⁵⁵ for long-range van der Waals interactions. Subsequent MD production runs of 500 ns were conducted at a temperature of 310 K and 1 bar using a semi-isotropic Parrinello-Rahman barostat. The final MD trajectories were analyzed and visualized using VMD⁵⁶ software, where the ligand RMSD calculations were also performed.

Data availability

The atomic coordinates for BQ3020–ET_B–miniG_s/q, ET1–ET_B–miniG_s/q, ET1–ET_A–miniG_s/q, Fab₃₀₁–macitentan–ET_A, Fab₃₀₁–ambrisentan–ET_A and Fab₃₀₁–zibotentan–ET_A complexes have been deposited in the Protein Data Bank (PDB) with the accession codes 8XVE, 8XVH, 8XVI, 8XVJ, 8XVK and 8XVL, respectively. The EM maps for BQ3020–ET_B–miniG_s/q, ET1–ET_B–miniG_s/q, ET1–ET_A–miniG_s/q, Fab₃₀₁–macitentan–ET_A, Fab₃₀₁–ambrisentan–ET_A and Fab₃₀₁–zibotentan–ET_A complexes have been deposited in the Electron Microscopy Data Bank (EMDB) with the codes EMD-38702, EMD-38704, EMD-38705, EMD-38706, EMD-38707 and EMD-38708, respectively.

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Acknowledgements

This work was supported by the National Key R&D Program of China (2022YFA1302902 to T.H.), the National Natural Science Foundation of China (32230026 to Z. Liu, 32271262 to T.H. and 82070085 to G.T.), the CAS Strategic Priority Research Program (XDB37030104 to Z. Liu), the Natural Science Foundation of Shanghai (21ZR1412900 to Y.S.), the Research Project of Shanghai Municipal Health Commission (20234Z0010 to G.T.). Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine at ShanghaiTech University. We thank the Shanghai Municipal Government and ShanghaiTech University for financial support. The cryo-EM data were collected at the Bio-Electron Microscopy Facility of ShanghaiTech University, with the assistance from Qianqian Sun, Dandan Liu. We also thank the staff at the Assay, Cell Expression, Cloning and Purification Core Facilities of iHuman Institute for their support.

Author information

These authors contributed equally: Junyi Hou, Shenhui Liu, Xiaodan Zhang, Guowei Tu.

Authors and Affiliations

Cardiac Intensive Care Center, Zhongshan Hospital, Fudan University, Shanghai, China
Junyi Hou, Guowei Tu, Yijie Zhang & Zhe Luo
iHuman Institute, ShanghaiTech University, Shanghai, China
Shenhui Liu, Xiaodan Zhang, Lijie Wu, Junlin Liu, Longquan Jiang, Qiwen Tan, Zhijie Liu & Tian Hua
School of Life Science and Technology, ShanghaiTech University, Shanghai, China
Shenhui Liu, Xiaodan Zhang, Hao Yang, Xiangcheng Li, Longquan Jiang, Fang Bai, Zhijie Liu & Tian Hua
Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China
Hao Yang, Xiangcheng Li & Fang Bai
Department of Anesthesiology, Zhongshan Hospital, Fudan University; Cancer Center, Zhongshan Hospital, Fudan University, Shanghai, China
Changhong Miao
Shanghai Key Laboratory of Perioperative Stress and Protection, Shanghai, China
Changhong Miao
Department of Critical Care Medicine, Shanghai Xuhui Central Hospital, Zhongshan Xuhui Hospital, Fudan University, Shanghai, China
Zhe Luo
Shanghai Key Lab of Pulmonary Inflammation and Injury, Shanghai, China
Zhe Luo

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Junyi Hou
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Shenhui Liu
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Xiaodan Zhang
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Guowei Tu
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Lijie Wu
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Yijie Zhang
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Hao Yang
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Xiangcheng Li
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Junlin Liu
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Longquan Jiang
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Qiwen Tan
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Z. Luo, T.H., Z. Liu and C.M. conceived and supervised the overall project. J.H. and S.L. purified the protein complex and prepared the samples for cryo-EM analysis. S.L., J.H., and L.W. collected cryo-EM data and performed cryo-EM map calculation, model building and the structure refinement under the supervision of Z. Liu and T.H. X.Z. performed the functional studies supervised by T.H. Y.Z. assisted with the functional studies. J.H. and Q.T. generated the ET insect expression constructs. H.Y. and X.L. performed the AlphaFold2 multimer prediction for Fab₃₀₁ supervised by F.B. J.L. and L.J. performed protein expression. Z. Liu, T.H., Z. Luo, C.M. S.L., J.H., and X.Z. analyzed the complex structures and designed structure-function experiments. T.H., G.T., J.H., and S.L. drafted the manuscript and designed the figures and tables. T.H. wrote the manuscript with the input from Z. Liu, Z. Luo, C.M. and all other authors.

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Correspondence to Zhijie Liu, Changhong Miao, Tian Hua or Zhe Luo.

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Hou, J., Liu, S., Zhang, X. et al. Structural basis of antagonist selectivity in endothelin receptors. Cell Discov 10, 79 (2024). https://doi.org/10.1038/s41421-024-00705-9

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Received: 26 February 2024
Accepted: 30 June 2024
Published: 30 July 2024
DOI: https://doi.org/10.1038/s41421-024-00705-9