Structural basis of ligand recognition and activation of the histamine receptor family

Zhang, Xuan; Liu, Guibing; Zhong, Ya-Ni; Zhang, Ru; Yang, Chuan-Cheng; Niu, Canyang; Pu, Xuanyu; Sun, Jingjing; Zhang, Tianyao; Yang, Lejin; Zhang, Chao; Li, Xiu; Shen, Xinyuan; Xiao, Peng; Sun, Jin-Peng; Gong, Weimin

doi:10.1038/s41467-024-52585-y

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Published: 27 September 2024

Structural basis of ligand recognition and activation of the histamine receptor family

Nature Communications volume 15, Article number: 8296 (2024) Cite this article

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Abstract

Histamine is a biogenic amine that is critical in various physiological and pathophysiological processes, including but not limited to allergic reactions, wakefulness, gastric acid secretion and neurotransmission. Here, we determine 9 cryo-electron microscopy (cryo-EM) structures of the 4 histamine receptors in complex with four different G protein subtypes, with endogenous or synthetic agonists bound. Inside the ligand pocket, we identify key motifs for the recognition of histamine, the distinct binding orientations of histamine and three subpockets that facilitate the design of specific ligands. In addition, we also identify key residues responsible for the selectivity of immethridine. Moreover, we reveal distinct structural features as determinants of Gq vs. Gs or Gs vs. Gi coupling differences among the histamine receptors. Our study provides a structural framework for understanding the ligand recognition and G protein coupling of all 4 histamine receptors, which may facilitate the rational design of ligands targeting these receptors.

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Introduction

Histamine is a widely distributed biogenic amine that is a neurotransmitter in the central nervous system and an important signaling molecule in the immune system, gut, and skin^1,2. Histamine is an important metabolite that is formed by L-histidine decarboxylase (HDC), which catalyzes the decarboxylation of L-histidine^2,3. Histamine is released most often by mast cells and basophils via degranulation in response to various immune and nonimmune stimuli^3,4,5. Moreover, it mediates a wide range of physiological processes, including but not limited to allergic reactions, inflammation, wakefulness, gastric acid secretion, and neurotransmission^6,7,8,9. Many histamine functions are mediated by G protein-coupled histamine receptors, which include four members, H1R, H2R, H3R, and H4R^{7,10,11,12,13}. Compared to other G protein-coupled receptors, histamine receptors have low sequence homology, which varies between 16-35% at the protein level¹⁴. Drugs targeting H1R, H2R, and H3R have been used to treat allergies, gastroduodenal ulcers, and narcolepsy^15,16,17. Indeed, the inflammatory response caused by histamine release has long been thought to be mediated by the histamine H1 receptor and H1 receptor antagonists, often referred to as antihistamines, have been used for years to treat allergies¹⁸. Commonly used histamine H1 receptor antagonists, such as diphenhydramine, promethazine, and chlorpheniramine, are the most widely used nonspecific antiallergic drugs¹⁹. Moreover, H2 antagonists, such as cimetidine, nizatidine, and ranitidine, are widely used to inhibit the oversecretion of gastric acid for the treatment of peptic acid diseases, including duodenal ulcers, gastric ulcers, gastroesophageal reflux and heartburn^20,21. In addition, the clinical use of the H3R antagonist pitrizant for narcolepsy (Waxx®) and obstructive sleep apnea (Ozawade®) has been approved.

Histamine receptors participate in various biological processes by binding to diverse G protein subtypes. For example, H1R is known to activate phospholipase C through the Gq/11 signaling pathway, resulting in increased intracellular DAG, IP3, and Ca²⁺ concentrations²². Notably, this signaling plays important roles in H1R-mediated atherosclerosis²³, capillary permeability²⁴, and airway obstruction²⁵ during asthma. After activation, H2R is known to couple to both the Gs and Gq proteins to stimulate gastric acid secretion, alleviate inflammatory responses^{26,27,28,29,30}, and enhance cardiac output and coronary artery blood flow³¹. In contrast to H1R and H2R, H3R and H4R primarily couple with Gi/Go proteins. Whereas H3R is known to modulate neurotransmitter release, appetite, and circadian rhythm via Gi/Go coupling^30,32,33, H4R is mostly expressed in the immune system and participates in eosinophil chemotaxis and degranulation and leukotriene production in mast cells^34,35,36. The structures of inactive H1R, H2R, and H3R, as well as the active state of H1R and H4R, have been recently elucidated^{37,38,39,40,41}. However, there is a lack of structural information regarding the active states of H2R and H3R, as well as agonist recognition, selectivity, and diverse G protein coupling mechanisms.

In this work, we determine nine cryogenic electron microscopy (cryo-EM) structures of the four histamine family receptor subtypes in complex with four different downstream G proteins in the presence of endogenous histamine or other synthetic ligands at resolutions ranging from 2.30 to 3.0 Å. The combination of structural analysis and pharmacological characterization enable us to provide important mechanistic insights into endogenous histamine recognition, synthetic agonist selectivity, receptor activation and diverse G protein coupling profiles within the histamine receptor family.

Results

Structural determination

To understand the molecular mechanism of agonist recognition in the histamine receptor (HR) family, we determined nine cryo-EM structures of the four histamine receptor subtypes in complex with four downstream G protein transducers (Gs, Gq, Gi, and Go) in the presence of endogenous ligand histamine or three synthesized agonists, including imetit, immepip, and immethridine, with an overall resolution ranging from 2.30 to 3.0 Å (Fig. 1, Supplementary Figs. 1–4, and Supplementary Tables 1, 2). The EM density maps enabled us to model the ligands, most receptor residues, and heterotrimeric G proteins. The N-terminus, C-terminus, and ICL3 of the four histamine receptors and the α-helix domain of the Gα subunit were not modeled because of poor EM densities (Supplementary Figs. 5, 6).

**Fig. 1: Overall structures of histamine receptors in complex with respective G proteins.**

A common motif for histamine recognition

The binding pockets of all four histamine receptors (H1R–H4R) encompassing the putative cryo-EM density of histamine were flanked by TM3 and TM5-TM7 segments. Among the 14 residues constituting the binding pockets of the four histamine receptors responsible for histamine recognition, five residues were structurally equivalent (Fig. 2a). Importantly, in histamine-bound H1R-Gq, H2R-Gs/q, H3R-Gi, and H4R-Gi complexes, an acidic residue D^3.32 plays a pivotal role in contacting histamine as described in many GPCRs in recognizing biogenic monoamines. Specifically, in the structures of the histamine-bound H1R-Gq and H2R-Gs/q complexes, the positively charged nitrogen in the ethylamine chain of histamine forms electrostatic interactions with D^3.32, while in H3R-Gi and H4R-Gi complexes, D^3.32 forms a hydrogen bond with the delta nitrogen in the imidazole ring of histamine, and this interaction is potentially further strengthened by the electrostatic interaction due to the potential protonated status of the delta nitrogen (Fig. 2b). Moreover, two hydrophobic residues, Y/V^3.33 and Y^6.51 of all HRs, formed interactions with the endogenous agonist histamine (Fig. 2b). These three residues, which are highly conserved in the histamine receptor family, constitute the D^3.32-Y/V^3.33-Y^6.51 motif that is critical for histamine recognition (Fig. 2a and Supplementary Fig. 7a–d). Consistent with these observations, mutations of D^3.32A completely impaired the activation of the four histamine receptors in response to stimulation with histamine (Fig. 2c and Supplementary Fig. 7a–d). These three residues, which are highly conserved in the histamine receptor family, constitute the D^3.32-Y/V^3.33-Y^6.51 motif that is critical for histamine recognition. However, it’s worth noting that the functional significance of this motif may vary between subtypes, such as H1R and H2R versus H3R and H4R, due to differences in binding modes. For H1R and H2R, D^3.32 and Y^6.51 are critical for histamine recognition, as the hydrogen bond contacts. For H3R and H4R, D^3.32 is necessary for histamine recognition.

**Fig. 2: Common interaction motif for histamine recognition.**

Distinct endogenous histamine binding orientation

Interestingly, the orientation of histamine binding to the ligand pockets of H3R and H4R is opposite to that observed with of H1R and H2R, resulting from the rotation of the imidazole ring by ~180° (Fig. 3a). The observed histamine binding modes were further supported by computational molecular dynamics (MD) simulation (Fig. 3b and Supplementary Fig. 8). Notably, two conserved aromatic amino acids in TM7, specifically F^7.39 and W^7.43, exclusive to H3R and H4R, are replaced by smaller hydrophobic residues I/L^7.39 and Y^7.43 in H1R and H2R (Fig. 3a, c). F^7.39 and W^7.43 in H3R and H4R formed strong π-π packings with the imidazole ring and thus could be responsible for the placement of the imidazole ring close to the TM7 of H3R and H4R. Consistent with this hypothesis, mutations such as F^7.39A and W^7.43A significantly reduced the activation of H3R/H4R in response to histamine stimulation (Fig. 3c, d). In contrast, two phenylalanines, F^6.52 and F^6.55 in TM6, which were conserved in H1R and H2R but replaced by T/S^6.52 and M/T^6.55 in H3R and H4R, formed strong π-π interactions with the imidazole ring of histamine in the H1R and H2R structures and could determine the specific orientation of the imidazole ring in the TM5 and TM6 of H1R and H2R (Fig. 3a, c). Additionally, we have generated the I/L7.39A and Y7.43A mutants in H1R (I454^7.39A, and Y458^7.43A) and H2R (L274^7.39A and Y278^7.43A), respectively. Together with the F7.39A and W7.43A corresponding position mutants in H3R (F398^7.39A and W402^7.43A) and H4R (F344^7.39A and W348^7.43A), we examined the effect of mutations at positions 7.39 and 7.43 on the binding affinities of histamine within the histamine receptor families (H1R–H4R) using radioligand binding assays. Consistent with the hypothesis, the mutations of F398^7.39A and W402^7.43A in H3R and F344^7.39A and W348^7.43A in H4R reduced the binding affinities of histamine by more than 30-fold. In contrast, the mutations of I/L454^7.39A and Y458^7.43A in H1R and L274^7.39A and Y278^7.43A in H2R had a significantly lesser impact on binding affinities, with reductions around 3-5 times, respectively. This is consistent with our structural observation that the F^7.39 and W^7.43 in H3R and H4R form pi-pi stacking with the imidazole ring of histamine, whereas in H1R and H2R, the corresponding residues at positions 7.39 and 7.43 do not form pi-pi interactions with the ethylamine chain of histamine. Thus, mutations at these residues have a lesser impact on the binding affinity of histamine to H1R and H2R (Supplementary Fig. 9). Collectively, we speculated that the specific presence of large hydrophobic residue motifs in TM6 and TM7, including the conserved F^6.52X₂F^6.55 in the TM6 of H1R and H2R and F^7.39X₃W^7.43 in the TM7 of H3R and H4R, are determinants of the specific orientation of the histamine imidazole ring.

Our findings contrast with those reported in a recently published study, where histamine was observed to bind to H4R in a manner similar to that seen in H1R/H2R⁴². Importantly, our histamine-bound H4R structures are coupled with heterotrimeric Gi proteins, whereas those resolved by Im et al. involve complexes with Gq. This distinction suggests the possibility that the varying binding modes of histamine to H4R may be influenced by the interaction with different downstream G proteins.

Ligand pocket of H1R and H2R

Structural comparison revealed that the ligand pockets of H1R and H2R are clearly different from those of H3R and H4R. The ligand pockets of H1R and H2R can be divided into two portions: the imidazole group recognition pocket (orthosteric pocket) and the secondary binding pocket (SBP) (Fig. 4a, b and Supplementary Fig. 10a, b). On the other hand, the ligand pockets of H3R and H4R predominantly consist of three portions: the orthosteric pocket, the SBP, and the transmembrane subpocket (TMSP), which is composed of seven residues (Fig. 4a, b).

Approximately 8 residues constitute the orthosteric pocket of H1R and H2R for histamine recognition, of which five residues are nearly identical (Fig. 4b, c). Importantly, the conserved hydrophobic residues in TM6 and TM7, including W^6.48, Y^6.51, F^6.52, and F^6.55, formed extensive hydrophobic interactions with the imidazole ring (Fig. 4a). In addition, T^3.37 formed a polar interaction with the amine group of the imidazole ring in H1R and H2R. In H1R, W158^4.56 and N198^5.46 formed hydrophobic and polar interactions with the imidazole ring, respectively (Supplementary Fig. 10c). Conversely, in H2R, D186^5.42 formed a charge‒charge interaction with histamine, which was not observed in H1R (Fig. 4a, b and Supplementary Fig. 10d).

Ligand pocket of H3R

In the four H3R complex structures, the ligands histamine, imetit, immethridine, and proxyfan contain imidazole groups with modified tails. In all these structures, the imidazole group is placed in the same position, highlighting the conserved mechanism of imidazole ring recognition in different agonists by H3R (Figs. 4a, 5a). Along with the extended tail length, the ligand pocket gradually moves toward the TMSP (Figs. 4a, b, 5a, b). In total, four hydrophobic residues from TM6 and TM7 and one polar residue from TM3 constituted the orthosteric pocket, in which F398^7.39 and W402^7.43 form π-π packings with the imidazole group, while Y374^6.51 and L401^7.42 form hydrophobic interactions with these four ligands (Figs. 4a, b, 5a, b). Specifically, D114^3.32 forms both hydrogen bonds and charge‒charge interactions with the nitrogen atom of the imidazole group (Figs. 4a, 5a). Consistent with these observations, the D114^3.32A mutation totally abolished the activation of H3R in response to stimulation with these agonists (Fig. 2c).

The presence of additional chemical groups in the agonists imetit, immethridine, and proxyfan revealed that the TMSP is composed of residues from TM3-TM6, which include Y115^3.33, C118^3.36, Y167^4.57, S203^5.43, E206^5.46, W371^6.48 and T375^6.52 (Fig. 5a, b). Along with the increase in the ligand tail length, then number of ligand interactions within the pocket also increases. For instance, the tail of histamine only interacts with Y115^3.33, C118^3.36, and E206^5.46, whereas the tail of proxyfan interacts with all seven residues in the TMSP (Fig. 5a, b). Similarly, this TMSP exists in H4R and is composed of eight residues from TM3-TM6 (Fig. 5a, b). The different imidazole ring binding orientations and the presence of the TMSP are unique to H3R and H4R, and these characteristics may assist the design of ligands that preferentially target these two histamine receptors over H1R and H2R.

Mechanism of immethridine selectivity for H3R

In contrast to immepip, which has high affinities for both H3R and H4R, immethridine showed selectivity toward H3R by approximately 300 times that of H4R (Fig. 6a). Compared with that of immepip, the piperidine ring of immethridine was replaced by a pridine ring, which was more rigid (Fig. 6b). Structural comparison of the immethridine-H3R-Go complex and the immepip-H4R-Gi complex structures indicated that the piperidine ring assumed a distorted conformation to accommodate Q347^7.42 in the immepip-H4R-Gi complex structure, whereas Q347^7.42 is not compatible with the flat conformation of the pridine ring in immethridine (Figs. 5a, 6b, c and Supplementary Fig. 11a, b). Notably, Q347^7.42 in H4R was replaced by L401^7.42 in H3R (Fig. 6b–d), which formed a suitable hydrophobic interaction with the pyridine ring (Supplementary Fig. 11a, b). The binding poses and interactions of immethridine and immepip within the ligand pockets of H3R and H4R was further confirmed by MD simulation, respectively (Supplementary Fig. 11c, d). Consistent with these observations, the L401^7.42 Q mutation of H3R decreased immethridine activity toward H3R by approximately 400-fold, whereas the Q347^7.42L mutation significantly increased the immethridine activity toward H4R. In contrast, the L401Q mutation of H3R and the Q347L mutation of H4R had little difference in the effect of immepip (Fig. 6a). Collectively, our structural and pharmacological analysis indicated that the specific interaction between the rigid pyridine ring of immethridine and L401^7.42 in H3R is a key determinant of immethridine selectivity for H3R over H4R. The sequence difference between L401^7.42 in H3R and Q347^7.42 in H4R could be utilized for the design of ligands that specifically target either H3R or H4R.

Activation of histamine receptors

Comparing the structure of active H1R with the previously published structures of inactive H1R⁴³, there is a dramatic outward movement of the intracellular part of TM6 (~27.4°) (Fig. 7a). In the extracellular region, there is an inward movement of TM6 (~13.1°) and TM3 (~12.1°) (Fig. 7a). On the cytoplasmic side, H1R contains the conserved D124^3.49R125^3.50 Y210^5.58 (DRY) motif and the N464^7.49 P465^7.50 XXF/Y468^7.53 motif. These motifs are substantially rearranged in active H1R relative to inactive H1R (PDB: 3RZE) (Fig. 7b, c). Moreover, we found that both the F424^6.44 and W428^6.48 residues in H1R moved downward and that W428^6.48 directly contacted histamine (Fig. 7d, e). Structural rearrangements of these two aromatic residues during receptor activation have been reported⁴⁴. A comparison of the three active state structures of H1R and H2R suggested that the conformations of F^6.44 and W^6.48 are identical in these two receptors. Similarly, in the active structure of H2R, W^6.48 also directly contacted histamine (Fig. 7e). In contrast, the conformations of W^6.48 in H3R and H4R are different from those in the other two receptors and do not directly interact with ligands (Fig. 7d, e and Supplementary Fig. 12a).

ICL3 is a structural determinant for the selective coupling of H2R to Gs and Gq

It has been reported that H2R couples with multiple downstream effectors upon activation, including Gs and Gq. We conducted a detailed exploration of both the Gs and Gq coupling mechanisms of H2R through structural analyses of H2R-Gs and H2R-Gq complexes (Fig. 8a). To assist in determining of H2R-Gq complex, a chimeric Gαsq was employed instead of Gαq (Supplementary Fig. 1d). Among 11 interface contacting residues between Gαsq and H2R, ten residues were identical to those in the Gαq subunit, and one residue differed. Consequently, we replaced these differing residues in Gαsq with the original residues in Gαq and performed MD simulation (Supplementary Fig. 12d, e). The computational simulation suggested that all the aforementioned residues formed interfaces with H2R, with additional contacts formed between Gαq and two residues in H2R (Fig. 8b). All of these interactions were further validated by mutagenesis analysis (Supplementary Fig. 15b). A total of 22 residues spanning TM3, TM5, TM6, Helix 8 (H8), and ICL2 and ICL3 of H2R were observed to form direct interactions with both the Gs and Gq trimers (Fig. 8b). There are more interactions observed in H2R-Gs complex than in the H2R-Gq complex, including additional contacts mediated by ICL2, ICL3, TM5, TM6, and H8 (Fig. 8b). Conversely, in comparison to Gs, Gq exhibited extra contacts with TM6 and a unique contact with TM7 of H2R (Fig. 8b).

**Fig. 8: ICL3, determinant for selective coupling of H2R to Gs and Gq.**

In total, 22 residues that interact with both Gs and Gq were derived from the TM3, ICL2, TM5, ICL3, TM6 and H8 of H2R (Fig. 8b). Three residues from TM3 (R116^3.50, A119^3.53, and V120^3.54), two residues from ICL2 (P123^ICL2 and L124^ICL2), and five residues from TM5–TM6 (I205^5.61, V208^5.64, A209^5.65, K231^6.32, and L236^6.37) were common in both H2R-Gs and H2R-Gq complex structures, and one residue from H8 (R293^8.48) of H2R form conserved hydrophobic packing with Gq and Gs (Supplementary Fig. 12b, c). In addition, Q212^5.68 of H2R formed H-bonds with Q350 of Gαq or R385 of Gαs (Fig. 8C). R215^5.71 of H2R formed conserved charge-charge interactions with both Gαq and Gαs (Fig. 8c). We speculated that these common interactions are determinants of H2R coupling to both Gs and Gq subtypes. Notably, N357 of Gαq formed a polar interaction with the backbone carbonyl oxygen of L291^7.56 of H2R, which is unique to H2R-Gq but absent in H2R-Gs (Supplementary Fig. 12f). Moreover, K223^6.24 and T226^6.27 formed additional H-bonds with D315 and the backbone carbonyl oxygen of S320 of Gαq, respectively (Supplementary Fig. 12g). Consistently, Mutations of K223^6.24A and T226^6.27A had a larger impact on Gq activation than Gs activation in response to histamine stimulation, consistent with these observations (Supplementary Fig. 15b).

Compared with Gαq, Gαs formed more residue contacts with H2R (Fig. 8b). For instance, V128^ICL2 of H2R formed extra hydrophobic interaction with A39 of Gαs (Supplementary Fig. 12h). In addition, A213^5.69 and K223^6.24 in TM5 and TM6 are responsible for more hydrophobic interactions with L394 and T350 of Gαs (Supplementary Fig. 12i). Moreover, R296^8.51 of H2R formed additional charge-charge interaction with E392 of Gαs. Interestingly, compared with that in the H2R-Gαq complex structure, the EM density of ICL3 of H2R in the histamine-H2R-Gs structure is more continuous and clear. Therefore, we modeled ICL3 in the histamine-H2R-Gs structure and analyzed its interaction with Gs. W222 in ICL3 of H2R forms a hydrogen bond and a cation-π interaction with T350 and R347 of Gαs, respectively (Fig. 8e). In our computationally simulated H2R-Gq complex, we observed a swinging motion of W222 (Fig. 8d), resulting in the complete abolition of the specific interactions between W222 and D315, as well as P318 in Gαq (D315 and P318 are the equivalent residues of R347 and T350 in Gαs) (Fig. 8e). Consistent with this observation, mutations of W222A and W222V totally abrogated the Gs activity of H2R in response to histamine stimulation, whereas the same mutations showed no significant effects on histamine-induced Gq activity downstream of H2R (Fig. 8f, g). Moreover, we speculated that ICL3 may play an important role in the selectivity of Gs and Gq. The important interaction between these two residues and Gαs is essential to stabilize ICL3 of H2R. Additionally, we compared the outward displacement of TM6 when the H2R receptor couples with Gq or Gs. Consistent with recent reports, we observed that TM6 exhibits a greater outward displacement for receptor coupling to Gs than to Gq⁴⁵ (Supplementary Fig. 13).

Determinants of Gs and Gi/o coupling differences among different histamine receptors

Whereas histamine receptors H1R and H2R primarily couple to Gs and Gq, H3R and H4R primarily couple with Gi/o. Structural and mutational analysis of H2R-Gs and H2R-Gq revealed that ICL3 is a potential structural determinant for the selective coupling of H2R to Gs and Gq (Fig. 8). An intriguing follow-up question is how different histamine receptor members selectively couple to Gs or Gi/Go proteins. Comparing the structures of H2R-Gs, H3R-Gi/o, and H4R-Gi, we found that six residues of TM3, TM5, and TM6 of H2R, H3R, and H4R were observed to form conserved interactions with both Gs and Gi (Fig. 9a, b). In addition, five residues of ICL2, TM6, and H8 of H2R, H3R, and H4R formed almost identical interactions with both Gs and Gi (Fig. 9b).

**Fig. 9: Determinants of Gs and Gi/o coupling differences between HRs.**

Interestingly, we found that the extended TM5 in H2R migrates toward TM6 starting from the R204^5.60 position by ~15° (Fig. 9c and Supplementary Fig. 14a), allowing substantially more interactions between the TM5 of H2R and Gs than the interactions between the TM5 of H3R or H4R and the Gi/Go proteins (Fig. 9a). Specifically, the H2R-Gs complex exhibited four residues from the TM5 (I205^5.61, V208^5.64, A213^5.69, and I227^6.28) of H2R that formed more hydrophobic packings with three residues of the α5 helix (L394, L393, Q384) of Gαs (Supplementary Fig. 14b). Moreover, Q212^5.68 and R215^5.71 of H2R formed H-bonds or charge‒charge interactions with Q385 and D381 from the α5 helix of Gαs, respectively (Supplementary Fig. 14c). In addition, I216^5.72 and W222^6.23 of H2R formed hydrophobic packing with Gs (Supplementary Fig. 14d). These interactions were observed only in H2R-Gs complex structures but not in structures of H3R/H4R in complex with Gi/Go proteins. Consistent with these observations, R215^5.71A and I227^6.28A mutations significantly decreased Gs coupling to H2R (Supplementary Fig. 12c). In contrast, L^5.71A and S/R^6.28A mutations of structurally equivalent residues in H3R/H4R showed no significant or fewer effects on H3R/H4R coupling (Supplementary Fig. 15c).

Structural comparison of H3R/H4R-Go/Gi structures with the H2R-Gs structure also revealed special structural features of these two receptors responsible for Gi/Go engagements. Unique to H3R, N^2.39, Q^34.57, and R^4.41 formed specific interactions with Gi/Go (Supplementary Fig. 14e). Moreover, common to both H3R and H4R, R^34.54, F/V^6.25 and R^6.29 specifically formed interactions with the α5 helix and α4-β6 turn of Gi/Go (Supplementary Fig. 14f–h). These interactions were not observed in H2R-Gs complex structures. Consistently, mutations of R^34.54 F/V^6.25 and R^6.29 residues significantly decreased histamine and their synthetic agonists induced Gi/Go activity of H3R or H4R (Fig. 9d and Supplementary Fig. 15a). Altogether, our studies suggested that the rotation and more compact packing of the TM5 in H2R with Gs and R^34.54-F/V^6.25-R^6.29 interactions of H3R and H4R with Gi/Go are determinants for the Gs and Gi/o coupling differences among different histamine receptor members.

Discussion

In summary, we have provided a structural landscape of ligand recognition and G protein subtype coupling of histamine receptors by determining 9 structures of the four receptors (H1R–H4R) in complex with four different G protein transducers (Gs, Gq, Gi, and Go) and bound by the endogenous ligand histamine or three other synthesized agonists. Analysis of these structures identified that the presence of the D^3.32-Y/V^3.33-Y^6.51 motif was shared by all histamine receptors, suggesting that this motif plays critical roles in histamine recognition. Interestingly, we observed that the endogenous agonist histamine binds to the ligand pocket of the four histamine receptors in different orientations, which results from the rotation of the imidazole ring by ~180^o. Further structural and sequence comparisons and mutational studies enabled us to identify that the conserved F^6.52X₂F^6.55 in the TM6 of H1R and H2R and F^7.39X₃W^7.43 in the TM7 of H3R and H4R are determinants of these distinct orientations. Detailed characterization of histamine binding to H1R and H2R, combined with a structural comparison with the ligand pocket of H3R and H4R to accommodate synthetic agonists, revealed that two specific pockets (orthosteric pocket and SBP) and three specific pockets (orthosteric pocket, SBP and TMSP) were present in H1R and H2R or H3R and H4R, respectively. Residue differences in these pockets may be exploited for the design of specific ligands targeting selected HR members. For example, the substitution of L401^7.42 in H3R by Q347^7.42 in H4R was the determinant of immethridine selectivity of H3R compared with H4R. Below the ligand pocket, we identified conformational changes in the key structural elements underlying the activation mechanism of H1R. On the cytoplasmic side, we found that ICL3 of H2R is the determinant of Gs/Gq coupling differences. Moreover, more compact packing of TM5 of H2R with Gs and the unique interactions of the R^34.54-F/V^6.25-R^6.29 motif of H3R and H4R with Gi/Go underlies Gs and Gi/o selectivity among the different histamine receptor members. This information may not only further our understanding of the ligand recognition, activation, and G protein coupling mechanisms of the histamine receptor family but may also be helpful for designing selective ligands targeting histamine receptor family members.

Methods

Constructs

DNA sequences encoding human histamine receptors (H1R, H2R, H3R, H4R) were codon-optimized and synthesized for Sf9 insect cell expression and cloned into a pFastBac vector containing an N-terminal bovine prolactin signal peptide followed by a FLAG epitope (DYKDDDD) and an 8×His tag. A fragment of β₂AR N-terminal tail (BN)⁴⁶ was introduced to improve protein expression. One copy of BN for H1R and H3R, and two copies for H2R and H4R were fused at the N-terminus of the receptors with a TEV protease cleavage site. A NanoBiT tethering strategy was used to improve sample quality as described previously⁴⁷. The LgBiT subunit was fused at the C-terminus of the receptors in different manners (Supplementary Fig. 1a). For H3R, LgBiT was directly fused at the C-terminus. For H1R, LgBiT was fused with a 15-amino acid linker (GSSGGGGSGGGGSSG). For H2R and H4R, residues after A308 and Q378 were truncated respectively and LgBiT was fused with the 15-amino acid linker.

A dominant negative human Gαi1 with four mutations (S47N, G203A, E245A, A326S)⁴⁸, miniGαo⁴⁹, miniGαs with two extra mutations (G226A and A366S), and miniGαq⁵⁰ were cloned into the pFastBac vector. Human Gβ1 with an N-terminal 6×His tag and human Gγ2 were cloned into the pFastBac-Dual vector. The HiBiT subunit was fused to Gβ1 at the C-terminus with a 15-amino acid linker (GSSGGGGSGGGGSSG).

Protein expression and purification

Nb35 and scFv16 were expressed and purified as previously described in refs. ^51,52. Briefly, Nb35 was expressed in BL21 (DE3) E.coli cells and purified by Ni affinity chromatography with a C-terminal 6×His tag. scFv16 was expressed in Tni insect cells and purified from the medium by Ni affinity chromatography before the C-terminal 8×His tag was removed by TEV protease (NEB, Cat# P8112S). Nb35 and scFv16 were further purified by size-exclusion chromatography and stored at −80 °C before use.

Histamine receptor-G protein complexes were co-expressed in Sf9 insect cells using the Bac-to-Bac method. Cells were infected with three types of viruses (receptor, Gα and Gβγ) at the ratio of 1:1:1 and cultured for 48 h. Cells were harvested by centrifugation and stored at −80 °C before use.

Purification of the complexes followed a common set of procedures. Frozen cells were thawed in lysis buffer containing 20 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 5 mM CaCl₂, 25 mU/ml Apyrase (NEB, Cat# M0398S), 1–10 μM agonist, 2.5 μg/ml leupeptin (Sigma-Aldrich, Cat# L2884), 300 μg/ml benzamidine (Sigma-Aldrich, Cat# B6506), 100 μM TCEP (Thermo Fisher, CAS 5961-85-3). The solution was incubated at room temperature for 2 h to facilitate complex formation. After incubation, the sample was centrifuged at 30,700×g for 30 min to collect cell membranes. The membranes were resuspended with a glass dounce homogenizer in solubilization buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 0.5% (w/v) LMNG, 0.1% (w/v) CHS, 10% (v/v) glycerol, 10 mM MgCl₂, 5 mM CaCl₂, 12.5 mU/ml Apyrase, 1–10 μM agonist, 2.5 μg/ml leupeptin, 300 μg/ml benzamidine, 100 μM TCEP and incubated at 4 °C for 2 h. The supernatant was isolated by centrifugation at 38,900×g for 45 min, and then incubated with Ni resin at 4 °C for 2 h. After binding, the resin was washed with 20 column volumes of buffer A consisting of 20 mM HEPES pH 7.5, 100 mM NaCl, 0.05% (w/v) LMNG (Anatrace, Cat# NG310), 0.01% (w/v) CHS (Sigma-Aldrich, Cat# C6512), 1–10 μM agonist, 2.5 μg/ml leupeptin, 300 μg/ml benzamidine, 100 μM TCEP with 20 mM imidazole before bound material was eluted with 5 column volumes of buffer A containing 400 mM imidazole. The eluate was supplemented with 2 mM CaCl₂ and incubated with M1 anti-FLAG resin overnight at 4 °C. The resin was washed with 10 column volumes of buffer A supplemented with 2 mM CaCl₂, and the protein was eluted with 3.5 column volumes of buffer A containing 5 mM EDTA and 200 μg/ml FLAG peptide. scFv16 was added to the eluate at the molar ratio of 1.3:1 over the receptor-G protein complex. After incubated on ice for 20 min, the sample was concentrated to 0.5 ml with a 100 kD cut-off centrifugal filter and loaded onto Superdex 200 10/300 GL column pre-equilibrated with buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% (w/v) GDN, 0.00015% (w/v) CHS, 1–10 μM agonist and 100 μM TCEP. Fractions of the monomeric peak were analyzed by SDS-PAGE and Coomassie staining. Then the desired fractions were pooled and concentrated for cryo-EM studies.

Histamine was used at the final concentration of 10 μM for H1R and H2R in the process of purification, but used at 1 μM for H3R and H4R. Imetit, Immepip, Immethridine, and Proxyfan were used at the final concentration of 1 μM. For the purification of the histamine-H2R-miniGs-Nb35 complex, scFv16 was not added, and Nb35 was added to the lysis buffer at a final concentration of 10 μg/ml. For the purification of the histamine-H2R-miniGq-scFv16 complex, the Superdex 200 column was substituted with a Superose 6 Increase 10/300 GL column to separate the monomeric complex from aggregation.

Cryo-EM sample preparation and data acquisition

For cryo-EM grids preparation of the H1R-Gq, H2R-Gq, H2R-Gs, H3R-Gi, H3R-Go, and H4R-Gi complexes, 3 μl of the purified complexes at ~20 mg/ml were applied onto glow-discharged holey carbon grids (Quantifoil, Au300 R1.2/1.3). Grids were plunge-frozen in liquid ethane using Vitrobot Mark IV (Thermo Fischer Scientific). Cryo-EM imaging was performed on a Titan Krios electron microscope at 300 kV accelerating voltage using a Gatan K3 Summit direct electron detector with an energy filter. Micrographs were collected with a nominal magnification of 81,000× using the EPU software in super-resolution mode at a calibrated pixel size of 1.07 Å and a defocus range of −1.2 to −2.2 μm. Each stack was acquired with an exposure time of 3.5 s and dose-fractionated to 32 frames with a total dose of 55 e^-Å⁻². A total of 6208 movies, 5078 movies, 5934 movies, 4222 + 2189 movies, 4382 movies, 5122 movies, 4925 + 1303 movies, 5021 movies, and 4464 movies were collected for Histamine-H1R-Gq, Histamine-H2R-Gq, Histamine-H2R-Gs, Histamine-H3R-Gi, Imetit-H3R-Gi, Proxyfan-H3R-Go, Immethridine-H3R-Go, Immepip-H4-Gi complex, and Histamine-H4-Gi complexes, respectively.

Cryo-EM data processing

Image stacks were subjected to beam-induced motion correction using MotionCor2⁵³. Contrast transfer function (CTF) parameters were estimated from motion-corrected images using Gctf⁵⁴.

For the datasets of Histamine-H1R-Gq complex, total of 8,895,197 particles were auto-picked using RELION 3.1⁵⁵ and then subjected to reference-free 2D classification to discard bad particles. After several rounds of 3D classification, two well-defined subsets were selected. Further 3D classification focusing the alignment on the complex, produced one high-quality class accounting for 91,716 particles. These particles were subsequently subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a map with an indicated global resolution of 2.66 Å at a Fourier shell correlation (FSC) of 0.143.

For the Histamine-H2R-Gq complex and Histamine-H2R-Gs complex, a total of 6,586,815 and 8,349,077 particles were auto-picked and then subjected to reference-free 2D classification to discard poorly particles, respectively. For the Histamine-H2R-Gq complex, after several rounds of 3D classification, one well-defined subset was selected. Further 3D classification focusing the alignment on the complex, produced one high-quality subset accounting for 118,539 particles. The selected subset was subsequently subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a map with an indicated global resolution of 2.97 Å at a Fourier shell correlation (FSC) of 0.143. For the Histamine-H2R-Gs complex, after several rounds of 3D classification, two well-defined subsets were selected. Further 3D classification focusing the alignment on the receptor, produced one high-quality subset accounting for 229,186 particles. These particles were subsequently subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a map with an indicated global resolution of 2.56 Å at a Fourier shell correlation (FSC) of 0.143.

For Histamine-H3R-Gi, Imetit-H3R-Gi, Proxyfan-H3R-Go, and Immethridine-H3R-Go complexes, a total of 10,166,561, 6,019,821, 7,515,408, and 7,271,515 particles were auto-picked using RELION 3.1 and then subjected to reference-free 2D classification to discard badly defined particles, respectively. For the Histamine-H3R-Gi complex, after several rounds of 3D classification, four well-defined subsets were selected. Further 3D classification focusing the alignment on the receptor, produced one high-quality class accounting for 319,658 particles. These particles were subsequently subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a map with an indicated global resolution of 2.70 Å at a Fourier shell correlation (FSC) of 0.143. For the Imetit-H3R-Gi complex, after several rounds of 3D classification, two well-defined subsets were selected. Further 3D classification focusing the alignment on the complex, produced one high-quality class accounting for 103,738 particles. The selected subset was subsequently subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a map with an indicated global resolution of 2.77 Å at a Fourier shell correlation (FSC) of 0.143. For the Proxyfan-H3R-Go complex, after several rounds of 3D classification, one subset was selected. Further 3D classification focusing the alignment on the complex, produced one well-defined class accounting for 85,007 particles. These particles were subsequently subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a map with an indicated global resolution of 2.77 Å at a Fourier shell correlation (FSC) of 0.143. For the Immethridine-H3R-Go complex, after several rounds of 3D classification, two subsets were selected. Further 3D classification focusing the alignment on the complex, produced one class accounting for 160,325 particles. The selected subset was subsequently subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a map with an indicated global resolution of 2.77 Å at a Fourier shell correlation (FSC) of 0.143.

For the Immepip-H4-Gi complex and the Histamine-H4-Gi complex, a total of 7,742,393 and 6,851,148 particles were auto-picked and then subjected to reference-free 2D classification to discard bad particles, respectively. For the Immepip-H4-Gi complex, after several rounds of 3D classification, one well-defined class was selected. Further 3D classification focusing the alignment on the complex, produced one high-quality subset accounting for 76,732 particles. The selected subset was subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a map with an indicated global resolution of 2.63 Å at a Fourier shell correlation (FSC) of 0.143. For the Histamine-H4-Gi complex, after several rounds of 3D classification, one subset was selected. Further 3D classification focusing the alignment on the complex, produced one well-defined subset accounting for 170,852 particles. The selected subset was subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a map with an indicated global resolution of 2.30 Å at a Fourier shell correlation (FSC) of 0.143.

Model building and refinement

The models were built based on previously reported structures. Briefly, coordinates of the subunits from reported structures were docked into the density map using UCSF Chimera. Then the model was manually built-in COOT and refined by real-space refinement using PHENIX. First, we built the model of histamine-H4R-Gi-scFv16 based on the M1R crystal structure (PDB: 5CXV) and the Gi-scFv16 crystal structure (PDB: 6CRK). Then, this model was used as a template for the histamine-H3R-Gi-scFv16 complex. For the apo-H3R-miniGo-scFv16 complex, miniGαo derived from the GPR97-miniGo complex (PDB: 7D77) was used to substitute the Gαi subunit before manual adjustment and refinement. These models were docked into the EM maps of other complexes with different ligands and refined with the ligands replaced. For histamine-H1R-miniGq-scFv16 complex, coordinates from antagonist bound H1R crystal structure (PDB: 3RZE), miniGαq from HT2A-miniGq-scFv16 complex (PDB: 6WHA) and Gβγ-scFv16 from histamine-H4R-Gi-scFv16 complex were docked into the EM density to generate an initial model. Coordinates of H1R in the final model was used as a template for H2R in histamine-H2R-miniGs-Nb35 complex, and miniGαs-Nb35 from D1R-miniGs-Nb35 complex (PDB: 7JVQ) and Gβγ from histamine-H4R-Gi-scFv16 complex were combined to complete the initial model. Coordinates of H1R in histamine-H1R-miniGq-scFv16 complex was substituted with refined H2R to generate the initial model for histamine-H2R-miniGq-scFv16. Restraints of all ligands were generated by eLBOW in PHENIX. The model statistics was validated using MolProbity and the details are provided in Supplementary Tables 1, 2. The extent of model overfitting was evaluated by refining the final model against one half-map. FSC curves were calculated between the resulting model and the half map as well as between the resulting model and the other half map for cross-validation.

G-protein dissociation assay

G-protein bioluminescence resonance energy transfer (BRET) probes, including Gα-Rluc (Gαs-Rluc, Gαi-Rluc and Gαq-Rluc, Gβ and Gγ-GFP and the Gα- Gβγ dissociation assay were generated and performed as described. In Gα- Gβγ dissociation assays, Gα-Rluc, Gβ, and Gγ-GFP were used as G-protein BRET probes. HEK293 cells were co-transfected with HR or mutants and the G-protein probes, then plated at 5 × 10⁴ cells per well in 96-well microplates. After a 24-h incubation at 37 °C, cells were washed with Tyrode’s buffer and stimulated with specified ligands at different concentrations. BRET signals were measured using coelenterazine 400a (5 μM) and a Mithras LB940 microplate reader with BRET filter sets. The BRET signal was calculated as the 515 nm/410 nm light emission ratio. HEK293 cells were regularly tested for mycoplasma contamination.

Enzyme-linked immunosorbent assay (ELISA)

The cell surface expression levels of target receptors were assessed by ELISA for G-protein dissociation assay. HEK293 cells, transfected with wild-type Histamine receptor or its mutants, were plated at 1 × 10⁵ cells per well in 24-well plates and incubated for 16 h at 37 °C with 5% CO₂. After fixation with 4% paraformaldehyde (w/v) and blocking with 5% BSA (w/v), the cells were incubated with an anti-FLAG primary antibody (1:1000 dilution) at 4 °C for 16 h and then with a secondary goat anti-mouse antibody (1:5000 dilution) for 1 h at room temperature. Following washing, a TMB (3,3’,5,5’-tetramethylbenzidine) solution initiated a color reaction, stopped by 0.25 M HCl. Optical density at 450 nm was measured using a TECAN luminescence counter (Infinite M200 Pro NanoQuant) and correlated with transfection plasmid amounts to determine expression levels. HEK293 cells were regularly tested for mycoplasma contamination.

Molecular dynamics simulation

To evaluate the binding stability of individual ligands within their corresponding Histamine receptor systems, including histamine-H1R, histamine-H2R, histamine-H3R, histamine-H4R, immethridine-H3R, and immepip-H4R, the molecular dynamics (MD) simulations were performed as previously described⁵⁶. When setting up the MD simulation systems, the protonation states of ionizable residues at pH 7.5 were determined using Gromacs2019.6, which features a built-in protonation prediction analysis function set as a default parameter. All MD input files were generated by the CHARMMGUI website, where the force field parameters of ligands were generated by the CHARMM General Force Field (CGenFF)⁵⁷. The structures used for MD simulations were embedded into a pre-equilibrated and periodic structure of 1-palmitoyl-2-oleoyl-snglycero-3-phosphatidylcholinemembrane. Subsequently, 0.15 M NaCl was added to balance the charge of the MD structures system using the Monte Carlo Method⁵⁸. Next, the systems were solvated into periodic hexagonal type TIP3P water box with dimensions of ~80 Å by 80 Å by 80 Å using replacement methods, utilizing the membrane orientation calculated by the Orientations of Proteins in Membranes database. The MD structures systems were first processed for energy minimization for 10,000 steps using the gmx mdrun module in Gromacs2019.6⁵⁹, with the first 5000 steps performed using the steepest descent method and the remaining 5000 steps using the conjugated gradient method. All MD structures systems were heated from 0 to 310 K in the NVT ensemble (number of particles, volume, and temperature are conserved) for more than 1000 ps. Next, production simulations were run at 1 atm in the NPT ensemble (number of particles, pressure, and temperature are conserved) for more than 1000 ps with 10.0 kcal mol⁻¹ Å⁻² harmonic restraints. The NVT ensemble, NPT ensemble, and the 200 ns MD production with a time step of 2 fs were performed using Gromacs2019.6. All analyses were completed in Gromacs2019.6 and VMD⁶⁰.

To examine the effect of the four residues Phe^6.52, Phe^6.55, Ile^7.39, and Tyr^7.43 on histamine recognition in H1R, a model with histamine bind to a mutated H1R was generated by replacing residues Phe^6.52, Phe^6.55, Ile^7.39, and Tyr^7.43 in histamine-bound H1R with their equivalents in H3R: Thr, Met, Phe, and Trp, respectively, using COOT software. The resultant four-residues base-mutant-H1R (FFIY-mutant-H1R) model was further used for MD stimulation as described above. To further validate the histamine binding orientation in H4R, we conducted MD simulations using a Histamine-H4R model by placing histamine in the opposite orientation within H4R compared to its position in our resolved structure. All analyses were completed in Gromacs2019.6 and VMD⁶⁰.

To better understand the effect of W222 in ICL2 of H2R on Gs and Gq selectivity in H2R, we performed MD simulation using the histamine-bound H2R-Gs and a Chimera-generated H2R-Gq complex model, respectively. Due to the absence of ICL3 structure in the resolved H2R-Gq complex structure, we first performed homology modeling using SWISS-MODEL. The complete H2R receptor sequence was inputted, and the homology model was generated using the resolved structure of the ICL3-deficient H2R receptor as a template. We extracted the receptor region of the H2R model generated using SWISS-MODEL and the Gq region of the AT1R-Gq complex (PDB: 7F6G)⁶¹, and the H2R-Gq complex model for MD simulations was generated by Chimera. All the MD input files were generated, and all these MD simulation systems are executed using the molecular dynamics simulation parameters set at the beginning.

Radioligand binding assay

The human histamine receptors (H1R, H2R, H3R, and H4R) and their mutations were transfected into HEK293 cells for Radioligand binding assay. WT and mutant plasmids were transfected into HEK293 cells using polyethyleneimine (PEI) regent according to the manufacturer’s instructions, and cultured at 37 °C with 5% CO₂ for 48 h. The transfected cells were collected using the ice-cold lysis buffer of 50 mM HEPES (pH 7.4) and 2 mM EDTA. The cells were suspended vertically at 4 °C for 20 min and centrifuged at 500 ×g for 10 min. The supernatant was centrifuged at 30,000×g for 30 min at superspeed and ice-cold buffer containing 75 mM Tris (pH 7.4), 1 mM EDTA, and 12.5 mM MgCl₂. The total membrane surface protein expression was determined by Bradford protein assay. For the saturation binding assay, the cell membrane (~100 μg) was added with different concentrations of [³H]-Histamine and incubated in a shaking bed at 30°C for 1 h, and the Nonspecific binding curve was determined under the action of 50 μM ligands. The same amount of cell membrane was used for the competitive binding assay. On the basis of the 30 nM isotope ligand, a competitive ligand with gradient concentration was added and incubated in a shaking bed at 30 °C for 1 h. Incubation was in binding buffer [50 mM HEPES (pH 7.4), 10 mM MgCl₂, 1 mM EDTA, 0.1% protease-free BSA, and 0.01% ascorbic acid]. The cell membranes were filtered over GF/C glass microfiber filters (Whatman) presoaked in an ice-cold binding buffer of 75 mM tris (pH 7.4), 1 mM EDTA, and 12.5 mM MgCl₂ with 0.5% polyethylenimine, which using a 24-well Filter Mate harvester (PerkinElmer), and rinsing three times with 3 mL ice-cold binding buffer to remove free [³H]-Histamine. After drying the filters, 500 μL liquid scintillation was added, and the liquid scintillation count was performed on the PerkinElmer liquid scintillation counter. All data were analyzed by GraphPad Prism 9 (GraphPad Software Inc.). Each measurement was repeated in at least three independent experiments, each in triplicate.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The 3D cryo-EM density maps of Histamine receptors signaling complexes have been deposited in the Electron Microscopy Data Bank under the following accession codes: EMD-39412 (Histamine-H1R-miniGq), EMD-39413 (Histamine-H2R-miniGs), EMD-39414 (Histamine-H2R-miniGq), EMD-39415 (Histamine-H3R-Gi), EMD-39416 (Imetit-H3R-Gi), EMD-39417 (Immethridine-H3R-miniGo), EMD-39418 (Proxyfan-H3R-miniGo), EMD-39419 (Histamine-H4R-Gi), and EMD-39420 (Immepip-H4R-Gi). The atomic models have been deposited in the PDB databases under the following accession codes: 8YN2 (Histamine-H1R-miniGq), 8YN3 (Histamine-H2R-miniGs), 8YN4 (Histamine-H2R-miniGq), 8YN5 (Histamine-H3R-Gi), 8YN6 (Imetit-H3R-Gi), 8YN7 (Immethridine-H3R-miniGo), 8YN8 (Proxyfan-H3R-miniGo), 8YN9 (Histamine-H4R-Gi), 8YNA (Immepip -H4R-Gi). All the raw data from our molecular dynamic simulations have been uploaded to Figshare (https://doi.org/10.6084/m9.figshare.25886296.v1). The relevant raw data from each figure or table in the main manuscript and in the Supplementary Information are provided as a Source Data file. Source data are provided with this paper.

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Acknowledgements

We thank the Cryo-EM Center at the University of Science and Technology of China for the support of cryo-EM data collection. We thank Dr Yongxiang Gao for assistance with cryo-EM data collection. And we thank the Translational Medicine Core Facility of Advanced Medical Research Institute, Shandong University, for technical support. This work was supported by the Ministry of Science and Technology of China grant 2019YFA0904100 and the Natural Science Foundation of China grant T2221005 (both to W.G.), National Science Fund for Excellent Young Scholars (32222038 to P.X.), National Science Fund for Distinguished Young Scholars Grant (82425105 to J.-P.S.), National Key R&D Program of China (2019YFA0904200 to J.-P.S. and P.X.), and Shandong Provincial Natural Science Fund for Excellent Young Scholars (ZR2021YQ18 to P.X.).

Author information

Xuan Zhang
Present address: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, PA, 15261, USA
These authors contributed equally: Xuan Zhang, Guibing Liu, Ya-Ni Zhong, Ru Zhang, Chuan-Cheng Yang.

Authors and Affiliations

Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230026, China
Xuan Zhang, Guibing Liu, Xiu Li, Xinyuan Shen & Weimin Gong
Key Laboratory Experimental Teratology of the Ministry of Education and Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Cheeloo college of Medicine, Shandong University, Jinan, Shandong, 250012, China
Ya-Ni Zhong, Ru Zhang, Chuan-Cheng Yang, Canyang Niu, Xuanyu Pu, Jingjing Sun, Tianyao Zhang, Chao Zhang, Peng Xiao & Jin-Peng Sun
Advanced Medical Research Institute, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250012, China
Xuanyu Pu, Lejin Yang & Jin-Peng Sun
School of Pharmacy, Binzhou Medical University, Yantai, Shandong, 264003, China
Xuanyu Pu
Department of Psychology, Qilu Hospital of Shandong University, Jinan, Shandong, 250012, China
Lejin Yang
NHC Key Laboratory of Otorhinolaryngology, Qilu hospital and advanced Medical Research Institute, Meili Lake Translational Research Park, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250012, China
Jin-Peng Sun
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, 100191, China
Jin-Peng Sun

Authors

Xuan Zhang
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Guibing Liu
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Ya-Ni Zhong
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Ru Zhang
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Canyang Niu
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Tianyao Zhang
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Chao Zhang
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Xiu Li
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Xinyuan Shen
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Peng Xiao
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Contributions

W.G., J.-P.S., and P.X. conceived the project and designed the research with X.Z., G.L., Y.-N.Z., R.Z. and C.-C.Y., X.Z., and G.L. performed protein expression and purification studies, screened cryo-EM grids, collected cryo-EM data, and processed the data under the supervision of W.G. J.-P.S., Y.-N.Z., R.Z., X.L., and C.-C.Y. designed the G protein dissociation assay and analyzed results, X.P., J.S., T.Z., X.S., and L.Y. generated the HRs mutant constructs and performed the cell-based assays. R.Z., C.N., and C.Z. performed MD simulations under the guidance of P.X. and J.-P.S., Y.-N.Z., C.-C.Y., R.Z., and X.Z. finished all the figures under the supervision of P.X., J.-P.S., and W.G. J.-P.S., W.G., and P.X. wrote the manuscript, with input from all authors.

Corresponding authors

Correspondence to Xuan Zhang, Peng Xiao, Jin-Peng Sun or Weimin Gong.

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Nature Communications thanks Javier García-Nafría, So Iwata and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Zhang, X., Liu, G., Zhong, YN. et al. Structural basis of ligand recognition and activation of the histamine receptor family. Nat Commun 15, 8296 (2024). https://doi.org/10.1038/s41467-024-52585-y

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Received: 06 January 2024
Accepted: 12 September 2024
Published: 27 September 2024
DOI: https://doi.org/10.1038/s41467-024-52585-y

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