CysLT 2 R structure determination

To facilitate crystallization, human CysLT 2 R was modified by truncating N- and C-termini, inserting a thermostabilized apocytochrome b 562 RIL17 into the intracellular loop 3 (ICL3), and introducing three stabilizing point mutations18: W511.45V, D842.50N, and F1373.51Y (superscript refers to the Ballesteros–Weinstein GPCR residue numbering scheme19). The engineered receptor was crystallized in lipidic cubic phase (LCP)20 in complex with three antagonists: ONO-2570366 (cpd 11a) (2.4 Å resolution in two different space groups), ONO-2770372 (cpd 11b; 2.7 Å), and ONO-2080365 (cpd 11c; 2.7 Å) (Supplementary Figs. 1–4 and Supplementary Table 1). To validate the structures and probe the role of key residues, involved in ligand binding and receptor function, we conducted cell surface expression and IP 1 stimulation and inhibition assays with a set of 24 mutants (Supplementary Figs. 5 and 6 and Table 1).

Table 1 Signaling and cell surface expression data for CysLT 2 R. Full size table

Overall architecture of CysLT 2 R

All CysLT 2 R structures adopt the canonical seven-transmembrane helical bundle architecture (Fig. 1a) and are structurally similar to CysLT 1 R-pranlukast16 (Supplementary Table 2). Overall CysLT 2 R conformations are identical to each other (Supplementary Table 2), except for the structure with cpd 11c, which is described below. Our further analysis, therefore, is focused on the highest resolution CysLT 2 R-11a structure, unless noted otherwise. Extracellular loop 2 (ECL2) in CysLT 2 R is stabilized by the highly conserved disulfide bond21 between C1113.25 and C187ECL2. An additional disulfide bond is formed between C311.25 and C2797.27 (Fig. 1c). Notably, both TM1 and TM7 are about one helical turn shorter than in CysLT 1 R, resulting in a ~5 Å shift of ECL3 tip (Fig. 1b).

Fig. 1: Structure of CysLT 2 R. a Overall structure of CysLT 2 R-11a (C222 1 space group). b Structural superposition of CysLT 2 R-11a (blue; C222 1 space group) with CysLT 1 R-pranlukast (yellow). c Comparison of disulfide bridges between CysLT 1 R (yellow) and CysLT 2 R (blue). Comparison of functional motifs: NPxxY (d) P-I-F (e), and DRY (f). Membrane boundaries are shown as dashed lines in a and b. Full size image

As expected, CysLT 2 R structures with antagonists 11a and 11b are captured in a fully inactive state. Similar to inactive structures of CysLT 1 R and other receptors from the δ-branch of class A GPCRs, the P5.50-I3.40-F6.44 microswitch is found in a distinct conformation (Fig. 1e)16, previously associated with activation of receptors from other class A GPCR branches. The role of this microswitch in receptors from the δ-branch is apparently different and is likely linked to the substitution of the “toggle switch” W6.48 with F6.48, which prevents this microswitch from accessing its inactive conformation. The highly conserved D[E]R3.50Y motif, in which R3.50 is stabilized in an inactive conformation via a salt bridge with D[E]3.49, is replaced by VR3.50F in CysLT 2 R (Fig. 1f). As expected, restoring the canonical ionic lock by V1353.49D in CysLT 2 R decreases the potency of LTD 4 while increasing the potency of antagonists through stabilization of the inactive conformation (Table 1). Restoration of Y in the D[E]RY motif via F1373.51Y mutation, which is also present in the crystallized construct, has no effect on the potency of LTD 4 or antagonists. Similarly, the stabilizing mutation W511.45V in the crystallized construct has little effect on ligand binding and receptor signaling. Finally, the third crystallization construct mutation D842.50N, a known stabilizing mutation in the conserved in class A GPCRs sodium-binding pocket22,23,24, abolishes LTD 4 -stimulated IP 1 production in CysLT 2 R, similar to its effect in other receptors25. Likewise, N2977.45C in the sodium-binding pocket results in a complete loss of signaling activity. Mutating N3017.49D in the conserved NP7.50xxY motif (Fig. 1d) (NPLLY in CysLT 2 R; DPLLY in CysLT 1 R) stabilizes the sodium-binding pocket and thus reduces LTD 4 signaling potency 6-fold, while increasing receptor surface expression and E max (Table 1).

Interestingly, the CysLT 2 R-11c structure shows a different orientation of the Y2215.58 microswitch along with a distinct conformation of the intracellular part of TM6, shifted ~5 Å outward compared with other CysLT 2 R structures (Supplementary Fig. 7a). Both changes are consistent with a partially active-like GPCR state26, which, however, lacks key activation-related changes in TM7 and sodium pocket. Molecular dynamics (MD) simulations show that this state is distinct from both active and inactive states and highly dynamic (Supplementary Fig. 7b, c), suggesting that CysLT 2 R-11c likely represents an intermediate conformational state, selected and stabilized by the crystal lattice.

Unlike CysLT 1 R structures, all CysLT 2 R structures, except for the complex with cpd 11c, possess a well-resolved intracellular amphipathic helix 8 (H8) running parallel to the membrane (Fig. 1b). While the function of H8 is not fully understood, a mounting evidence points toward its importance in the regulation of G protein and β-arrestin binding27,28. Notably, the junction between TM7 and H8 in CysLT 1 R contains a rare GG8.48 motif, which likely increases dynamics of H8. On the other hand, position 8.48 in CysLT 2 R is occupied by E3108.48, which stabilizes the junction and the inactive state by forming salt bridges with R1363.50 and K2446.32 (Fig. 1f). Removing these interactions by E3108.48A or E3108.48G results in a slightly increased potency of LTD 4 in IP 1 signaling assays (Table 1).

Ligand-binding pocket and ligand-receptor interactions

In all CysLT 2 R structures, a strong electron density for the ligand (Supplementary Fig. 4) is present inside the central cavity of the receptor that consists of residues from all seven TMs and ECL2. It has a narrow opening (~3 Å diameter) between ECLs into the extracellular space and a larger access cleft (~5 Å across) from the lipid bilayer between TM4 and TM5 (Fig. 2a). All antagonists cocrystallized with CysLT 2 R share the same 3,4-dihydro-2H-1,4-benzoxazine scaffold and bind in the pocket in similar conformations (root mean square deviation < 0.3 Å in the common scaffold, Fig. 2). A key anchoring residue Y1193.33, conserved in CysLTRs, forms multiple polar contacts with the benzoxazine part, carboxylic group, and amide linker of all ligands (Fig. 2b–d). Y1193.33F mutant shows decreased potencies for both LTD 4 and antagonists in IP 1 assay (Table 1). The N-linked carboxypropyl moiety makes salt bridges with K371.31 and H2847.32 that are specific to CysLT 2 R. Mutating these residues to their CysLT 1 R counterparts (K371.31R or H2847.32Q) drastically decreases potencies for LTD 4 activation as well as inhibition by antagonists, suggesting distinct binding interactions of these ligands with CysLT 1 R and CysLT 2 R.

Fig. 2: Ligand-binding pocket of CysLT 2 R. a Sliced surface representation of the ligand-binding pocket in CysLT 2 R. b Binding pose of cpd 11a and details of ligand-receptor interactions. Schematic diagrams of CysLT 2 R interactions with cpds 11a and 11b (c) and cpd 11c (d). Residues are colored according to the effect of their mutations on the antagonist potency in IP 1 signaling assays: light red—strong effect, blue—no effect, white—not tested. The outline color indicates the effect of mutations on LTD 4 potency: red—strong effect, red dashed—nonresponsive mutants, blue—no effect. Full size image

The hydrophobic bottom part of the ligand-binding cleft containing the butoxybenzene group of cpd 11a is formed by side chains of TM3-TM5 and, in case of cpd 11c, extends to L1654.52 and I1664.53. Y1273.41 forms an interhelical hydrogen bond with the carbonyl oxygen of Val2085.45, stabilizing a Pro-induced kink in TM5 (Fig. 2c, d). An aromatic residue in position 3.41 at the intersection of TM3-TM5 was previously described to confer receptor stabilization29. Interestingly, mutation Y1273.41W slightly improves CysLT 2 R surface expression and potencies of LTD 4 and cpd 11c, however, dramatically decreases the potency of cpd 11a to inhibit LTD 4 -induced IP 1 accumulation, likely because of a clash between bulky tryptophan and 2-chloro-5-fluoro-phenyl group of cpd 11a. S1694.56 forms a hydrogen bond with the carbonyl group of L1654.52 and interacts with the fluorine atom of cpd 11c phenyl group. Mutation S1694.56A does not affect EC 50 for LTD 4 and IC 50 for cpd 11a but moderately improves inhibition by cpd 11c.

Antagonist selectivity to CysLTR subtypes

To understand the mechanism of ligand selectivity, we performed docking of 18 derivatives of the common 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid scaffold30,31 with a large spectrum of CysLT 1 R/CysLT 2 R selectivity (Supplementary Table 3). Docking models of the most selective compounds in this structure-activity relationship (SAR) series30, cpd 13e (1,800-fold selective for CysLT 1 R) and cpd 15b (200-fold selective for CysLT 2 R), are shown in Fig. 3, alongside with cpd 11a (dual CysLT 1 R/CysLT 2 R), cocrystallized with CysLT 2 R, and pranlukast (4,500-fold selective for CysLT 1 R as shown in Supplementary Fig. 6a), cocrystallized with CysLT 1 R.

Fig. 3: Structural determinants of antagonist selectivity to CysLTR subtypes. a Examples of compounds used in the docking studies, with their IC 50 values toward CysLT 1 R and CysLT 2 R shown in yellow and blue, respectively. IC 50 values for pranlukast were obtained in this work (3.8 ± 0.7 nM (CysLT 1 R) and ~17,000 ± 12,000 nM (CysLT 2 R), expressed as mean ± s.d. of three independent experiments, tested in quadruplicate) and for other ligands were quoted from ref. 30. The common 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid scaffold is shown in gray. Overview of the ligand-binding pocket with the docked ligands for CysLT 1 R (b) and CysLT 2 R (c). Inserts show docking poses and details of ligand interactions with CysLT 1 R and CysLT 2 R. Full size image

SAR analysis revealed that the most important factor for CysLT 2 R selectivity is the length of the alkyl chain for the O-substituents (R1), where longer phenylpentyl group in cpd 15b achieves much higher CysLT 2 R selectivity than phenylbutyl in cpd 11a, cpd 13e, and pranlukast or phenylpropyl in some other compounds such as cpd 15a (Supplementary Table 3). Comparison of the contacts of these substituents in CysLT 1 R-pranlukast and CysLT 2 R-11a suggests that in CysLT 1 R the cleft opening to the lipid membrane is restricted by a hydrophobic ridge formed by F1504.52, F1123.41, and V1965.45, while in CysLT 2 R the replacement F4.52L removes this restriction, making the cleft more open. Accordingly, docking of cpd 15b into CysLT 1 R results in a strained alkyl chain and a clash of the terminal phenyl group with F1504.52, while in CysLT 2 R the phenyl group readily extends outside of the cleft (Fig. 3b, c). Moreover, the phenylbutyl group in this and other scaffolds tolerates methyl and halogen decorations in the ortho and meta positions, which enables tuning pharmacological properties of the ligand such as solubility and stability, as exemplified by the development of gemilukast32.

SAR analysis of the N-substituent (R2) suggests that its length as well as the presence of a carboxyl group in this scaffold has critical influence on IC 50 values for both CysLTRs. Indeed, docking of cpd 13e, the most selective antagonist in this series, shows that the oxo-pentanoic-acid moiety of this ligand forms a hydrogen bond with Y261.35, while CysLT 2 R has F411.35 at this position and cannot form a hydrogen bond with the ligand (Fig. 3b, c). Further elongation of this derivative chain is limited by the size of this subpocket. Interestingly, removal of the carbonyl group, as in cpds 14a-c and 15b, shifts selectivity toward CysLT 2 R, suggesting that a flexible carboxy-alkyl chain is favored for this receptor30. Altogether, CysLT 1 R and CysLT 2 R crystal structures provide atomic level insights into the mechanisms of ligand recognition and subtype selectivity. This knowledge should contribute to the rational design of more efficient antagonists with improved affinity/efficacy or subtype selectivity profiles.

Structural insights into CysLT 2 R disease-related mutations

Finally, our structures provide rational explanations of the two most common disease-associated single-nucleotide variants (SNVs) in CysLT 2 R: M2015.38V, related to atopic asthma13,33, and the oncogenic L1293.43Q mutation34,35. M2015.38 together with M1724.59, L1734.60, and L1985.35 define the shape of the hydrophobic part of the ligand-binding pocket. Substitutions of L1985.35 with alanine or M2015.38 with alanine or leucine result in nonresponsive mutants that bind LTD 4 but fail to stimulate IP 1 production. In contrast to the alanine or leucine substitution, the atopic asthma-associated variant M2015.38V still responds to LTD 4 stimulation. However, this mutation significantly decreases LTD 4 potency and efficacy to induce IP 1 accumulation when compared with the wild-type CysLT 2 R (Table 1). These results along with a similar effect of N2025.39H suggest the importance of ligand-dependent TM5 displacement in CysLT 2 R activation. Indeed, all three TM5 residues (L1985.35, M2015.38, and N2025.39) that are important for potency interact with the benzamide core of antagonists, which distinguish them from agonists, and thus likely modulate TM5 conformation and dynamics that control activation (Fig. 4a).

Fig. 4: Naturally occurring missense SNVs, mapped on the CysLT 2 R structure. a M2015.38V polymorphism, associated with atopic asthma. b L1293.43Q mutation, related to uveal melanoma and blue nevi. c SNVs from the ExAC database and L1293.43, colored according to their location: ligand-binding pocket (red), microswitches (blue), sodium site (green), and G protein and β-arrestin-binding interface (yellow). Full size image

The second disease-relevant SNV, L1293.43Q, has been associated with uveal melanoma and blue nevi34,35,36. A hydrophobic amino acid is present in this position in 97% of class A receptors, most frequently L3.43 (73%), but also M3.43 as in CysLT 1 R. Located at the bottom of the sodium pocket, a large hydrophobic side chain in position 3.43 is part of a hydrophobic layer, which is important for stability of the inactive state. Mutation of L3.43 to a polar residue or to a small alanine residue can disrupt the hydrophobic layer (Fig. 4b, c), facilitating water and sodium passage37,38 and leading to receptor activation. Indeed, it was shown that mutation in position 3.43 to R, K, A, E, or Q induces constitutive activation in several receptors39, often resulting in distinct physiological disorders. In CysLT 2 R, we found that L1293.43Q displays constitutive activity for the G q pathway with a fourfold increase in basal IP 1 accumulation and is unresponsive to LTD 4 stimulation (Supplementary Fig. 6b, c).