Structural basis of the cysteinyl leukotriene receptor type 2 activation by LTD4

Significance

Our research addresses a significant gap in the current understanding of CysLT2R activation by its endogenous ligand, cysteinyl leukotriene D4 (LTD4). Utilizing cryo-electron microscopy, we present the structure of LTD4-bound human CysLT2R in complex with the Gαq protein. The structure reveals a previously uncharacterized spacious polar pocket and the lateral ligand access route into the orthosteric pocket between transmembrane helices 4 and 5. These findings illuminate the crucial role of transmembrane domain helix 3 in sensing agonist moieties. Our study not only advances the foundational understanding of CysLT2R activation but also provides a rational basis for the design of novel therapeutic agents targeting this receptor, an emerging therapeutic target with significant implications across inflammatory and oncological conditions.

Abstract

The G protein–coupled cysteinyl leukotriene receptor CysLT2R plays intricate roles in the physiology and pathogenesis of inflammation-related processes. It has garnered increasing attention as a potential therapeutic target for atopic asthma, brain injury, central nervous system disorders, and various types of cancer. In this study, we present the cryo-electron microscopy structure of the cysteinyl leukotriene D4 (LTD4)-bound human CysLT2R in complex with a Gα_q protein, adopting an active conformation at a resolution of 3.15 Å. The structure elucidates a spacious polar pocket designed to accommodate the two branched negative ends of LTD4 and reveals a lateral ligand access route into the orthosteric pocket located on transmembrane domain helix (TM) 4 and 5. Furthermore, our findings highlight the crucial role of transmembrane domain helix 3 in sensing agonist moieties, representing the pivotal mechanism of receptor activation for both CysLT1R and CysLT2R. Collectively, the insights derived from our structural investigation establish a foundation for comprehending CysLT2R activation by its endogenous ligand LTD4, offering a rational basis for the design of drugs targeting CysLT2R.

Cysteinyl leukotriene receptors (CysLTRs) are members of the class A G protein–coupled receptors (GPCRs), specifically categorized within the lipid receptor subfamily. They activate both Gα_q and Gα_i, but preferentially couple to Gα_q proteins to activate downstream signaling pathways (1–4). Within this receptor family, two primary subtypes, CysLT1R and CysLT2R, exist, distinguished by low sequence homology and distinct tissue distribution in vivo (4). The CysLT1 receptor is ubiquitously expressed in various human tissues, including the lungs, peripheral blood leukocytes, spleen, and placenta. In contrast, the CysLT2 receptor displays a unique expression pattern, notably in organs such as the heart, brain, and adrenal glands (5, 6). The distinct tissue distribution of these receptors, coupled with the identification of asthma-related polymorphisms in CysLT2R (7), suggests divergent roles for each receptor subtype in both physiological and pathological contexts. While the CysLT1 receptor is associated with a spectrum of respiratory diseases and proves to be an effective therapeutic target for conditions like asthma and allergic rhinitis (8), the CysLT2 receptor extends its influence beyond respiratory ailments to encompass cardiovascular, nervous system disorders, and cancer (9, 10). Numerous studies indicate the potential of CysLT2R as a novel therapeutic strategy against conditions such as cerebral ischemia stroke (11–13), poststroke depression (PSD) (14), inflammatory itch (15), and colorectal cancer (16, 17). These findings underscore the versatility of CysLT2R as a potential target across a range of pathological conditions.

Cysteine leukotrienes D4, C4, and E4 serve as the endogenous ligands for CysLTRs, originating from arachidonic acid through the action of 5-lipoxygenase (4). Despite their structural similarities, these ligands exhibit variations in their affinity for CysLTRs. Specifically, among them, LTD4 emerges as the preferred endogenous ligand for CysLT1R, while both LTD4 and LTC4 demonstrate comparable affinity for CysLT2R (Fig. 1A). Recognized as the potent bronchoconstrictor in the body, LTD4 plays a pivotal role in triggering both acute and chronic asthma (18). Moreover, LTD4 has been implicated in various inflammatory diseases, including inflammatory bowel disease, with elevated levels of cysteine leukotrienes detected in the gut of affected patients. Notably, the LTD4-CysLT2R signaling pathway assumes critical importance in colon cancer. In contrast to CysLT1R, activation of CysLT2R by LTD4 does not induce cell proliferation in colon cancer cells. Instead, CysLT2R exhibits heightened antitumor activity in intestinal epithelial cells, suggesting a distinctive role in counteracting tumor progression (16). This nuanced understanding of the differential effects of CysLT2R activation by LTD4 holds significant implications for unraveling the complexities of inflammatory and neoplastic processes in the colon.

Fig. 1.

Cryo-electron microscopy (EM) structure of the active-state CysLT2R–Gα_q–scFv16 complex. (A) Schematic representation of signal transduction between LTC4, LTD4, LTE4, and CysLT1/2R. (B) Cryo-EM density map of LTD4–CysLT2R–Gα_q complex. (C) Structural model determined after refinement in the cryo-EM map and LTD4 density map. LTD4 is displayed in salmon, LTD4-bound CysLT2R in medium aquamarine. The Gq heterotrimer is colored by subunits: Gα_q, medium purple; Gβ, dark goldenrod; Gγ, cornflower blue; scFv16, gray.

As an appealing drug target for conditions including allergic airway inflammation, inflammatory arthritis, and atherosclerosis, the development of antagonists targeting CysLTRs has been a focal point of research. Presently, antiasthma medications such as montelukast, zafirlukast, pranlukast, and others, which specifically target CysLT1R, are the only options available. The absence of medications targeting CysLT2R likely stems from the more intricate physiology and etiology associated with this receptor subtype. Despite extensive research, the complexities involved in targeting CysLT2R have hindered the development of therapeutic options. Numerous antagonists have been synthesized to date, including the dual antagonist BAYu9773 (19) the selective antagonist HAMI3379 (20), and BayCysLT2 (21). Notably, HAMI3379 has emerged as a widely employed tool compound and potential therapeutic drug for investigating the physiological functions of CysLT2R, as evidenced by its extensive use in studies (11, 14, 22, 23). The utilization of HAMI3379 underscores its significance in advancing our understanding of CysLT2R biology and its potential role as a therapeutic target in various pathological conditions.

The advancement of drug designs targeting CysLT2R has been impeded by the limited access to receptor activation mechanisms, a challenge exacerbated by the fact that most drug designs are engineered with endogenous ligands. Although the crystal structure of CysLT2R bound to its antagonists ONO-2570366, ONO-2080365, and ONO-2770372 was published in 2019 (24), the absence of CysLT2R structures bound to agonists has constrained our understanding of ligand recognition mechanisms and receptor activation. This knowledge gap hinders the development of more effective targeted drugs.

In addressing this limitation, we present the cryo-EM of human wild-type CysLT2R activated by the endogenous ligand LTD4. Our findings suggest that Extracellular Loop 2 (ECL2) covers the binding pocket and participates in ligand recognition, shedding light on the specific mechanism through which LTD4 activates the receptor. Complemented by mutagenesis studies, the structural insights we provide establish a foundation for understanding the intricate relationship between CysLT2R activation and the active conformation of the Gα_q protein. Furthermore, this information serves as a rational basis for the design of novel anticysteinyl leukotriene drugs, holding promise for future therapeutic developments in this domain.

Result

Overall Structure of the LTD4-Bound CysLT2R–Gα_q Complex.

To facilitate structure determination, human CysLT2R was modified by truncating C-terminus (1 to 337), inserting a thermostabilized apocytochrome b562RIL into the intracellular loop 3 (ICL3) (25), and introducing the NanoBiT tethering strategy to improve the stability and homogeneity of the complex (26). Additionally, a Tobacco etch virus (TEV) cutting site and maltose-binding protein (MBP) are introduced into the construct at the C-terminus for expression and purification. Concurrently, an engineered Gα_q chimera (27) was generated based on the mini-Gα_s/q71 (28) scaffold in which the N terminus was replaced by the corresponding Gα_i1 sequence. Unless otherwise stated, Gα_q refers to the engineered Gα_q used for structural determination. The engineered Gα_q can bind both Nb35 and scFv16 (29–31), but the LTD₄-bound CysLT2R–Gα_q structure only has visible density with scFv16 antibodies in the final EM density map. This was possible due to the weak occupation of Nb35 or the dynamic characteristics of Nb35 binding to the engineered Gα_q (32, 33).

Coexpression of the CysLT2 receptor and G protein occurred in Hi5 insect cells, followed by the purification of the CysLT2R–Gα_q complex to homogeneity for cryo-EM analysis using the Titan Krios microscope. The structure, resolved at a resolution of 3.15 Å (Fig. 1B and SI Appendix, Fig. S1), provided insights into the extracellular domains, transmembrane regions, and extracellular domains in complex with Gα_q, Gβ, Gγ, and scFv16. The majority of EM density maps were well resolved, enabling the unambiguous modeling of the complex, including the ligand LTD4, the Gα_q protein heterotrimer, scFv16, and the majority of amino acid side chains of the receptor CysLT2R (Fig. 1C). However, BRIL was absent from the map due to its high flexibility, a common outcome in other GPCR structure determinations (34). Additionally, the highly flexible N-terminal extracellular domain resulted in the nonresolution of the entire N-terminal residues (1 to 30) in the CysLT2R map. Similarly, the alkyl tail of LTD4 is too flexible and results in low density (Fig. 1C and SI Appendix, Fig. S2).

The LTD4-Binding Pocket of CysLT2R.

LTD4, 5[S]-hydroxy, 6[R]-S-cysteinyl-glycinyl-7,9-trans-11,14-cis-eicosatetraenoic acid Cysteine Residue (Cys) (Fig. 2A), a crucial metabolite originating from arachidonic acid and synthesized by leukocytes, assumes significance in various physiological processes. The CysLT2R structure delineated in this study represents a significant advancement, offering the insights into the recognition of the endogenous ligand LTD4. Similar to the docking study of the LTD4 into CysLT1R (35), LTD4 follows a comparable path and shares interactions with antagonists in both CysLTR receptors. The LTD4-binding pocket in our CysLT2R structure stretches from ECL2 across the receptor toward a gap between TM4 and TM5, deep in the middle section of the 7TM bundle (Fig. 2B). The orthosteric site in CysLT2R comprises two classic essential modules: a polar-network region at the top and a hydrophobic region deep in the cavity (Fig. 2C). Notably, this specific ligand-binding pocket pattern aligns with observations in other lipid receptor structures, including LTB4 (36), GPR119 (37), GPR84 (34), Lysophosphatidic Acid (LPA) receptors, and Sphingosine-1-Phosphate (S1P) receptors (38, 39), indicating a conserved architecture for accommodating endogenous ligands characterized by polar headgroups and acyl-tail hydrophobic frameworks. This structural congruence suggests commonalities in the binding motifs and recognition mechanisms shared by diverse lipid receptors.

Fig. 2.

LTD4-binding pocket of CysLT2R. (A) LTD4 chemical formula (Numbered by the number of base carbon atoms). Divided into three regions according to hydrophilic and hydrophobic, hydrophilic region (blue), hydrophobic region (yellow). (B) LTD4 (salmon) binding pocket of CysLT2R (medium aquamarine). The transmembrane helix 4-5 of CysLT2R and the cryo-EM density of LTD4 are highlighted. (C) Sliced surface representation of the LTD4-binding pocket in CysLT2R. Hydrophilic region (blue) and hydrophobic region (yellow). (D–G) Detail interactions between LTD4 and CysLT2R. C1 interactions (D); ECL2 interaction (E); C6 polar interactions (F); nonpolar interactions between the hydrophobic tail of LTD4 and the key residues from CysLT2R interactions (G). (H) Dose–response curves of LTD4 in activating CysLT2R with mutations in the binding pocket with IP one assay. The response data were normalized by WT receptor within each individual experiment. Data from three independent experiments, each of which was performed in triplicate, are presented as mean ± SEM. Detailed statistical evaluation is shown in SI Appendix, Table S1.

Distinctive to CysLT2R, the ligand pocket forms in the extracellular half of transmembrane helix 1-7 (TM1-7) and extracellular loop 2 (ECL2). The extracellular (EC) part of the binding pocket is aligned with polar and charged residues that engage in interactions with LTD4 (SI Appendix, Fig. S3). Notably, the middle segment of ECL2 deflects downward toward the orthosteric site below, enveloping the carboxylic acid group situated at the C1 position of LTD4. Y119^3.33 form a hydrogen bond with the C1 carboxylic acid group, stabilized by additional polar interactions between the C1 carboxylic acid group and the main chain of E189 (Fig. 2D). This configuration is additionally stabilized by two hydrogen bond interactions: one involving the side chain of Y98^2.64 from TM2 and the main chain carboxyl group of the L188^ECL2 and the other involving the side chain of K194^ECL2 and the main chain carboxyl group of the E189^ECL2. In addition, the extensive hydrophobic interactions between L190^ECL2 and L198^5.35 at the C-terminal of ECL2, complemented by a synergistic contribution from L271^6.59 also contribute to this configuration, underscoring the pivotal role of the ECL2 in LTD4 recognition (Fig. 2E and SI Appendix, Fig. S4). In contrast, the C6 cysteinyl-glycinyl group forms extensive polar interactions with the polar residues surrounding the outer rim of the pocket, which is open to the extracellular milieu. Specifically, the carboxylic acid group of the glycinyl forms hydrogen bond interactions with the side chains of K37^1.31, Y98^2.64, H270^6.58, and H284^7.32, which is further stabilized by the positive side chain of the residue R94^2.60 (Fig. 2F). Mutations in these residues markedly reduce or completely abolish LTD4 signaling (Fig. 2H and SI Appendix, Table S1). Additionally, the C1 carboxylic acid group forms a hydrogen bond with the side chain of carboxylic acid group of the LTD4 glycinyl, potentially contributing to the stabilization of the LTD4 recognition by CysLT2R (Fig. 2F). This detailed elucidation underscores the intricate network of polar interactions governing ligand recognition and receptor activation in the context of CysLT2R.

The hydrophobic ligand-binding cleft, responsible for coordinating the long alkyl chain of LTD4, can be distinctly partitioned into two segments. The upper section, featuring residues such as M172^4.59, L173^4.60, L198^5.35, and M201^5.38, primarily interacts with the middle segment of LTD4, securely enclosed within the transmembrane domain (TMD) core of CysLT2R (Fig. 2G). Concurrently, the lower part, comprising residues like L165^4.52, I166^4.53, I204^5.41, A205^5.42, and V208^5.45, firmly seizes the alkyl tail of LTD4, extending into the lipid bilayer through the gap between Transmembrane Helices 4 (TM4) and 5 (TM5). This distinctive binding feature corroborates that the opening between TM4 and TM5 can serve as a pivotal gate for lateral ligand entry into the orthosteric pocket, aligning with propositions from previous studies (35). Collectively, these detailed structural analyses provide indispensable insights, enriching our comprehension of the intricate mechanism governing LTD4 recognition by CysLT2R.

Activation Mechanism of CysLT2R.

The elucidated structure of Gα_q-coupled CysLT2R in complex with its endogenous agonist, LTD4, in conjunction with the previously reported antagonist-bound CysLT2R structure, has afforded an opportunity to probe the intricacies of agonist-mediated activation. A meticulous structural comparison, particularly with the antagonist ONO-2570366-bound CysLT2R (PDB code: 6RZ6), reveals subtle rearrangements at the extracellular ends of helix bundles. In contrast, marked conformational shifts manifest at the cytoplasmic end of the receptor, where TM5 and TM6 move outward by approximately ~4 Å (L229) and ~10 Å (V240), respectively (Fig. 3 A–C). Furthermore, the cytoplasmic TM7-Helix8 kink undergoes a distinct conformational shifting away from the TMD core. These structural alterations facilitate the coupling of the C-terminal helix of Gα_q to the receptor core cavity, thereby initiating the signaling cascade. This insightful structural analysis unveils the dynamic changes underlying the transition from an antagonist-bound to an agonist-bound state, shedding light on the molecular mechanism of agonist-mediated activation in Gα_q-coupled CysLT2R.

Fig. 3.

Activation mechanism of CysLT2R. (A–C) An overall comparison of LTD4-bound (receptor: medium aquamarine), ONO-2570366-bound (receptor: dark gray; PDB:6RZ6) CysLT2R. A 10 Å outward displacement LTD4-bound CysLT2R of TM6 was measured by the Cα of V6.28. Side view (A), extracellular view (B), and intercellular view (C). (D) Detailed comparison of interactions between LTD4-binding (LTD4: salmon) and ono-2570366 binding (ONO-2570366: cornflower blue; PDB:6RZ6) CysLT2R (The ligand transparency is reduced). (E) Toggle switch. (F) PIF motif (extracellular view). (G) VRF motif (extracellular view). (H) NPxxY motif. The movement of helices from inactive state to active state is highlighted with red arrows.

The structural analysis of CysLT2R bound to both agonist and antagonist reveals the pivotal role of TM3 in receptor activation. Notably, critical large residues within the ligand-binding pocket undergo significant rotamer changes upon agonist binding. Specifically, the key anchoring residue Y119^3.33, conserved in CysLTRs, forms multiple polar contacts with various components of ONO-2570366. In the active structure, the presence of the agonist LTD4 induces a major conformational change in the side chain of Y119^3.33, subsequently influencing the positions of M122^3.36, Y123^3.37, and I126^3.40, all shifting in the same direction (Fig. 3D and SI Appendix, Fig. S5 A–C). As a consequence, the hydrogen bond interaction between Y123^3.37 and H264^6.52 weakens, resulting in the downward shift of H264^6.52, which forms a van der Waals interactions with the toggle switch residue F260^6.48 (SI Appendix, Fig. S5 D and E). The combined rotamer changes of H264^6.52 and F260^6.48 from TM6, along with the displacements of Y119^3.33, M122^3.36, Y123^3.37, and I126^3.40 from TM3, contribute to a downward force on F256^6.44 of the conserved P^5.50I^3.40F^6.44 core triad (SI Appendix, Fig. S5F). As observed in the structures of active CysLT2R and inactive CysLT2R, F256^6.44 adopts distinct rotameric conformations, which cause notable rearrangement of the V^3.49R^3.50F^3.51 motif and NP^7.50xxY^7.53 motifs that function as microswitches (Fig. 3 E–G). Furthermore, the unique ionic lock formed between the E310^8.48 and the residues R136^3.50 and K244^6.32 in TM3 and TM6 becomes disrupted (SI Appendix, Fig. S6), subsequently releasing constraints on the relative movement of TM6 and creating more cavities for G protein binding. The distinct shift of the cytoplasmic TM7-Helix8 kink is accompanied by the rearrangement of TM6, in which residue Y305^7.53 points into the interior of the TM domain during the CysLT2R activation (Fig. 3H). Highlighting the conserved nature of these dynamic structural changes across the GPCR family, this intriguing behavior of conserved motifs mirrors observations in other family A G protein–coupled receptors (GPCRs) with available active- and inactive-state structural data.

Furthermore, the superposition of antagonist-bound CysLT1R and CysLT2R (PDB code: 6RZ5 for zafirlukast-bound CysLT1R and PDB code: 6RZ6 for ONO-2570366-bound CysLT2R), both in inactive states, reveals that these receptors adopt similar conformations of critical residues from TM3, including Y119^3.33, M122^3.36, Y123^3.37, and I126^3.40 (SI Appendix, Figs. S7 and S8A). Notably, the R121^3.50 of the F^3.49R^3.50C^3.51 motif in CysLT1R forms two hydrogen bond interactions with the main chain carboxyl groups of K230^6.32 and S298^7.56 in TM6 and TM7, stabilizing the inactive CysLT1R and precluding downstream transducer binding (SI Appendix, Fig. S8B). This is reminiscent of the ionic lock formed between the E310^8.48 and the residues R136^3.50 and K244^6.32 in TM3 and TM6 in inactive CysLT2R structure, suggesting that CysLT1R possibly employs a similar activation process as CysLT2R. Collectively, these observations indicate that TM3 may play conserved roles in receptor activation for both CysLT1R and CysLT2R.

The Gα_q-Binding Interface of CysLT₂R.

CysLT2R exhibits the capability to interact with various G proteins, with a pronounced preference for Gα_q. In typical class A GPCR–G protein complexes, the insertion of the C-terminal α5 helix of Gα_q into the cytoplasmic compartment formed by TMs, ICL2, and ICL3 facilitates signal transduction (Fig. 4A). The primary interaction site between CysLT2R and Gα_q occurs at the α5 interface, involving both polar and hydrophobic interactions. Specifically, R136^3.50 within the DRY motif forms a hydrogen bond with the N359 and Y358 backbone carbonyl group of Gα_q. Intriguingly, N311^8.49 on receptor Helix8 establishes a hydrogen bond with the side chain of N359 and an additional hydrogen bond with E357 backbone carbonyl group on Gα_q. Additionally, N359 on Gα_q forms a hydrogen bond with the backbone carbonyl groups of CysLT2R G309^8.47 (Fig. 4B). These polar interactions contribute to the stability of the binding of Gα_q to CysLT2R. The findings suggest that the TM7-Helix8 kink site serves as the key binding site of CysLT2R to Gα_q. Earlier studies have demonstrated the crucial role of ICL2 in influencing the coupling selectivity of Gα_q proteins (40). Specifically, an arginine or lysine base from ICL2 may interact polarly with the αN domain of Gα_q. However, in CysLT2R, the H148 of ICL2 does not establish similar polar interactions with the αN domain of Gα_q (Fig. 4C). Conversely, F144 of CysLT2R ICL2 engages in hydrophobic interactions with phenylalanine residues F196 and F343 at the αN–α5 interface of the Gα_q subunit, establishing an additional receptor–Gα_q interface (Fig. 4D). This may underscore the conserved hydrophobic residue of ICL2, playing a crucial role in the selection of Gα_q. Aligning the complex structures with reported class A GPCRs (M1R, H1R, 5HT_2AR) coupled to Gα_q/G₁₁ shows a substantial shift in the relative orientation of α5 and αN when configured as receptors. The Gα_q proteins in these receptor–Gα_q complexes have a similar clockwise rotation viewed from the intracellular to extracellular direction (SI Appendix, Fig. S9). Collectively, these findings suggest that the interaction between CysLT2R (TM7-H8 linker and ICL2) and Gα_q significantly contributes to the stabilization of the CysLT2R–Gα_q complex.

Fig. 4.

Gα_q coupling of CysLT2R. (A) Cytoplasmic cavity formed by TMs of CysLT2R, with the C-terminal α5 helix of the Gα_q inserted. (B) Polar interactions between CysLT2R and α5 helix of Gα_q. (C and D) Interaction sites between ICL2 and Gα_q.

Discussion

The cryo-EM structure presented in this study provides detailed insights into the activation mechanism of the CysLT2R by its endogenous ligand LTD4, emphasizing the pivotal role of ECL2 in ligand recognition. The ligand pocket accommodates the two branched negative ends of LTD4, involving extensive polar interactions with key residues. Notably, the carboxylic acid group at the C1 position of LTD4 forms a hydrogen bonds with residues Y119^3.33 and E189^ECL2, showcasing the importance of these interactions in ligand recognition. The intricate network of polar interactions, especially those mediated by residues Y98^2.64, K194^ECL2, and other polar residues in ECL2, underscores the specificity and stability of LTD4 binding to CysLT2R. In addition, the hydrophobic ligand-binding cleft, responsible for coordinating the long alkyl chain of LTD4, is detailed, revealing distinct segments that interact with different portions of the ligand. The upper section interacts with the middle segment of LTD4 within the transmembrane domain core, while the lower part firmly seizes the alkyl tail, extending into the lipid bilayer. This unique binding feature suggests a pivotal gate formed by TM4 and TM5 for lateral ligand entry into the orthosteric pocket, aligning with previous propositions.

The structural analysis of the LTD4-bound CysLT2R–Gα_q complex also reveals a complex network of interactions and conformational changes that underlie the activation process. A detailed comparison with the antagonist-bound CysLT2R structure elucidates subtle rearrangements at the extracellular ends of helix bundles, with marked conformational shifts observed at the cytoplasmic end. Specially, distinct rotameric conformations of key residues from TM3 cause notable rearrangement of the V^3.49R^3.50F^3.51 and NP^7.50xxY^7.53 motifs, which function as microswitches initiating the signaling cascade. The conserved nature of these dynamic structural changes across the GPCR family is highlighted, suggesting a common mechanism for receptor activation. The disruption of the ionic lock formed between E310^8.48 and residues in TM3 and TM6 creates additional cavities for G protein binding, further supporting the role of TM3 in receptor activation. The dynamic changes underlying the transition from an antagonist-bound to an agonist-bound state provide valuable insights into the molecular mechanism of agonist-mediated activation in Gα_q-coupled CysLT2R. In addition, comparisons with other leukotriene receptors, specifically CysLT1R, and their similar conformations of critical residues from TM3 suggest a conserved role of TM3 in receptor activation for both CysLT1R and CysLT2R. This observation adds depth to our understanding of the activation process and suggests commonalities in the activation mechanisms within the leukotriene receptor family.

One of the notable findings is the receptor’s ability to interact with various G proteins, with a clear preference for Gα_q. In typical class A GPCR-G protein complexes, the C-terminal α5 helix of Gα_q inserts into the cytoplasmic compartment, facilitating signal transduction. Our study reveals that the primary interaction site between CysLT2R and Gα_q occurs at the α5 interface, involving both polar and hydrophobic interactions. Specifically, key residues from the V^3.49R^3.50F^3.51 motifs establish hydrogen bonds with specific backbone carbonyl groups on Gα_q, contributing to the stability of the binding. This highlights the crucial role of the TM7-Helix8 kink site as the key binding site of CysLT2R to Gα_q. Previous studies have indicated the significance of ICL2 in influencing the coupling selectivity of Gα_q proteins, and our findings align with this notion. The hydrophobic interactions between specific residues on ICL2 of CysLT2R and Gα_q, especially F144 at position 34.51, form an additional receptor–Gα_q interface, potentially explaining the receptor’s ability to bind to both Gα_q and Gα_i with a preference for Gα_q.

In conclusion, the cryo-EM structure of the LTD4-bound CysLT2R–Gα_q complex provides a comprehensive understanding of the structural basis of CysLT2R activation by its endogenous ligand. The detailed elucidation of the ligand-binding pocket, activation mechanism, and Gα_q-binding interface enriches our knowledge of the molecular events governing leukotriene receptor activation. These findings lay a solid foundation for the rational design of drugs targeting CysLT2R, offering potential avenues for therapeutic interventions in diseases associated with dysregulated leukotriene signaling. The study contributes to the broader field of GPCR research by providing unique insights into the activation dynamics of this important class of receptors.

Materials and Methods

Construct Cloning.

The wild-type CysLT2R receptor (residues 1 to 337) was cloned into the pFastBac1 vector. The N terminus contains hemagglutinin signal peptide (HA) and heat-stable fusion protein BRIL, and the C terminus is partially fused to the LargeBit of NanoBiT, which can bind to the Gβ of the fused SmBiT, thus making the complex more stable. At the same time, tobacco acid virus (TEV) cleavage site and maltose binding protein (MBP) were introduced into the C terminus to promote protein expression and purification. In order to bind to both Nb35 and scFv16, a Gi1 N-terminal multifunctional chimera Gα_q was designed based on the mini-Gα_s backbone. Gα_q, rat Gβ1, and bovine Gγ2, as well as scfv16, were cloned into the pFastBac1 vector. All constructs were generated using the Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd.) and verified by DNA sequencing (Genomics).

Protein Complex Expression and Purification.

We used the Bac-to-Bac baculovirus system (Invitrogen) for expression in Trichoplusia ni (Hi5) insect cells. Cell cultures were grown in ESF 921 serum-free medium (Expression system) to a density of 3.2 × 10⁶ cells /mL. CysLT2R receptor, Gα_q, rat Gβ1, bovine Gγ2 and scfv16 were co-infected at a ratio of 1:1:1:1:1. After 48 h of infection, cells were harvested by centrifugation at 2,000 rpm (Thermo Fisher, H12000) for 20 min and frozen at −80 °C for further use.

For the purifications of LTD4-bound CysLT2R–Gα_q complex, cell pellets were thawed in 20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl₂, 5 mM CaCl₂, 10% glycerol, 20 μg/mL Nb35, 25 mU/mL apyrase (Sigma-Aldrich), and protease inhibitor cocktail (TargetMol). The suspensions were incubated for 1.5 h at room temperature. Subsequently, 0.5% (w/v) n-dodecyl-β-d-maltoside (DDM, Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate (CHS, Anatrace) were added to solubilize complexes for 3 h at 4 °C. The supernatant was collected by centrifugation at 30,000 rpm for 35 min and then incubated with dextrin resin (Dextrin Beads 6FF, Smart Life Sciences) at 4 °C for 3 h. After that, the resin was collected by centrifugation at 500 g for 10 min, loaded onto a gravity flow column and washed with 10 column volumes of buffer containing 20 mM HEPES pH7.4, 100 mM NaCl, 5 mM MgCl₂, 5 mM CaCl₂, 10% glycerol, 100 μM TCEP, 20 μg/mL Nb35, 0.05% (w/v) DDM and 0.01% (w/v) CHS. The detergent of washing buffer was then displaced by 0.1% (w/v) lauryl maltose neopentylglycol (LMNG, Anatrace) and 0.02% (w/v) CHS for 10 column volumes washing, followed by 0.03% (w/v) LMNG, 0.01% (w/v) glyco-diosgenin (GDN, Anatrace) and 0.008% (w/v) CHS for 10 column volumes washing. The protein was then treated with His-tagged TEV protease on column and further incubated at 4 °C for overnight. The elution was concentrated with an Amicon Ultra Centrifugal Filter (MWCO 100 kDa) and injected onto a Superose™ 6 Increase 10/300 GL (GE Healthcare) with running buffer 20 mM HEPES pH 7.4, 100 mM NaCl, 100 μM TCEP, 0.00075% (w/v) LMNG, 0.00025% (w/v) GDN and 0.0002% (w/v) CHS to separate complex from contaminants. Monomeric protein complexes of CysLT2R were collected and incubated with LTD4 for 2 h at 4 °C and then concentrated for cryo-electron microscopy experiments.

Expression and Purification of Nb35.

Nanobody-35 (Nb35) with an N-terminal pelB signal peptide and a C-terminal 6×His tag was expressed in the periplasm of Escherichia coli BL21(DE3) bacteria. Cultures were grown at 37 °C in LB media containing 50 μg/mL ampicillin to an OD600 of 1.0 and induced with 0.1mM IPTG at 28 °C, 180 rpm for 12 h. Cells were harvested by centrifugation (4,000 ×g, 30 min) and resuspended in buffer containing 20 mM HEPES pH 7.4, 500 mM NaCl and then centrifuged to remove cell debris. The supernatant was incubated with pre-equilibrated Nickel resin at 4 °C for 2 h. After washing with 20 column volumes of wash buffer containing 20 mM HEPES pH 7.4, 100 mM NaCl, 10% glycerol, Nb35 was eluted with wash buffer adding 300 mM imidazole. The protein was further purified by HiLoad 16/600 Superdex 75 column with 20 mM HEPES pH 7.4, 100 mM NaCl. Peak fractions were concentrated to 2 mg/mL with 15% glycerol and kept frozen at −80 °C for later use.

Cryo-EM Grid Preparation and Data Collection.

Three-microliter of the purified complex was applied onto a freshly glow-discharged Quantifoil holey carbon grid (R1.2/1.3, Cu/Rh, 300 mesh). Cryo-EM grids were prepared with the Vitrobot Mark IV plunger (FEI) set to 8 °C and 100% humidity. The sample was incubated for 10 s on the grids before blotting for 3.5 s (double-sided, blot force 1) and plunge-frozen in liquid ethane.

Data collections were performed on an FEI Titan Krios (FEI) electron microscopy, equipped with a Gatan K3 direct electron detection device, and operated at an acceleration voltage of 300 kV. Then, 5,534 movies were acquired at a nominal magnification of 105,000, corresponding to a pixel size 0.824 Å. Image acquisition was performed with EPU Software (FEI Eindhoven, Netherlands). We collected a total of 36 frames accumulating to a total dose of 50 e⁻ Å^-2 over 2.5 s exposure.

Image Processing and Map Construction.

MotionCor2 was used to perform the frame-based motion-correction algorithm to generate drift-corrected micrograph for further processing and defocus parameters were estimated by CTFFIND 4.0 (41, 42).

3D structure of TRH-TRHR G protein complex (PDB entry: 7WKD) previously resolved was used as references for automatic picking (43). The subsequent steps of particle picking, extraction, classification, and post processing of refined models were performed with Relion3.0 (44). A total of 4,654,998 particles were extracted from the cryo-EM micrographs. Then, 1,273,629 particles were remained after two rounds of reference-free two-dimensional (2D) classification. Mask three-dimensional (3D) classification on the receptor part was used to separate out 391,827 particles that resulted to a clearer density of CysLT2R (SI Appendix, Fig. S1).

Model Building and Refinement.

CysLT2R structure from Alphafold2 prediction was used as the starting reference model for receptor building (45). Structures of Gα_q, Gβ, Gγ, and the scFV16 derived from PDB entry 7WKD (43) were rigid body fit into the density using UCSF Chimera (46). The model was extensively modified using COOT (47) and PHENIX (48). The model was finalized by rebuilding in ISOLDE (49) followed by refinement in PHENIX with torsion-angle restraints to the input model. The final model statistics were validated using Comprehensive validation (cryo-EM) in PHENIX (48) and provided in SI Appendix, Table S2. All molecular graphics figures were prepared using Chimera7, Chimera X (50), and PyMOL (Schrödinger, LLC.).

Inositol Phosphate Accumulation Assay.

IP1 production was measured using the IP-One HTRF kit (Cisbio, 621PAPEJ). Briefly, 24 h after transfection, cells were harvested and resuspended in IP1 stimulation buffer at a density of 6 × 10⁶ cells/ml. Cells were then plated onto 384-well assay plates at 42,000 cells/7 µL/well. Another 7 µL IP1 stimulation buffer containing ligand was added to the cells, and the incubation lasted for 60 min at 37 °C. Then, 3 µL each of IP1-D2 and Ab-Crypt reagents were added and incubated for 1 h at RT. The plate was read on a EnVision multiplate reader using a HTRF (homogeneous time-resolved fluorescence) filter set (λex = 320 nm, λem = 620 and 655 nm).

Surface Expression Analysis.

Cell-surface expression for WT CysLT2R and mutants was monitored by a fluorescence-activated cell sorting (FACS) assay. In brief, AD293 cells expressing Flag-tagged CysLT2R were harvested 24 h after transfection. Cells were suspended by PBS buffer, then followed by the incubation with PE Mouse anti DDDDK-Tag mAb (ABclonal) at a dilution of 1:100 for 15 min at 4 °C, and then PBS buffer was added to cells. Finally, the surface expression of CysLT2R was monitored by detecting the fluorescent intensity of PE with Guava easyCyte 8HT (Merck Millipore).

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

The cryo-EM map of the LTD4–CysLT2R–Gα_q complex has been deposited in the Electron Microscopy Data Bank under accession codes: EMD-60980 (51), with the atomic coordinate deposited in the Protein Data Bank under accession codes: 9IXX (52). All other data are included in the manuscript and/or SI Appendix.