Ligand-binding Pocket Research Articles

Conservation of the cooling agent binding pocket within the TRPM subfamily.

Transient receptor potential (TRP) channels are a large and diverse family of tetrameric cation-selective channels that are activated by many different types of stimuli, including noxious heat or cold, organic ligands such as vanilloids or cooling agents, or intracellular Ca2+. Structures available for all subtypes of TRP channels reveal that the transmembrane domains are closely related despite their unique sensitivity to activating stimuli. Here, we use computational and electrophysiological approaches to explore the conservation of the cooling agent binding pocket identified within the S1-S4 domain of the Melastatin subfamily member TRPM8, the mammalian sensor of noxious cold, with other TRPM channel subtypes. We find that a subset of TRPM channels, including TRPM2, TRPM4, and TRPM5, contain pockets very similar to the cooling agent binding pocket in TRPM8. We then show how the cooling agent icilin modulates activation of mouse TRPM4 to intracellular Ca2+, enhancing the sensitivity of the channel to Ca2+ and diminishing outward-rectification to promote opening at negative voltages. Mutations known to promote or diminish activation of TRPM8 by cooling agents similarly alter activation of TRPM4 by icilin, suggesting that icilin binds to the cooling agent binding pocket to promote opening of the channel. These findings demonstrate that TRPM4 and TRPM8 channels share related ligand binding pockets that are allosterically coupled to opening of the pore.

Pyrethroids and reproductive function: some endocrine disrupting perspectives from molecular simulations.

Pyrethroids are widely used insecticides with huge applications for household as well as agricultural purposes and contribute to improved product quality and higher yields. In recent decades, the demand for pyrethroids has increased significantly due to advantages such as broad-spectrum efficacy, high insecticidal potential, and lower pest resistance. However, several studies have suggested that human exposure to pyrethroids leads to reproductive problems. Sex hormone-binding globulin (SHBG) is an important hormone transport protein regulating the availability of steroids at their target site. The aim of our study was to investigate the structural interactions of commonly used pyrethroids, cypermethrin and deltamethrin, with ligand binding pocket of SHBG. Cypermethrin and deltamethrin were docked into the steroid binding pocket of SHBG using Schrodinger's induced fit docking (IFD) followed by molecular dynamics (MD) simulation studies. The resultant SHBG-pyrethroid complexes from IFD experiments were subjected to structural analysis including the molecular interactions followed by binding energy estimation. The analysis revealed that both the ligands were tightly bound in the SHBG pocket with high percentage of commonality among the SHBG residues between the indicated pyrethroid ligands and the SHBG native ligand, dihydrotestosterone (DHT). The estimated binding energy values for cypermethrin were less but close to the values calculated for the SHBG native ligand, DHT. However, the estimated binding energy values for deltamethrin were higher compared to the values calculated for SHBG native ligand, DHT. Furthermore, the MD simulation results also revealed the higher stability of SHBG-deltamethrin than SHBG-cypermethrin complex. To sum up, the results suggested that deltamethrin has a greater capability than cypermethrin to prevent sex steroid hormone from binding to SHBG, even though both pyrethroids have this ability. Consequently, this might hamper the circulatory transport of sex steroid hormones and their availability at the target site, subsequently interfering with reproductive function.

Structure-based design and screening of hydrogel copolymer/Fe3O4 composite microspheres for magnetic solid phase extraction of bisphenol A from aqueous samples

It is of great significance to monitor bisphenol A (BPA) in the environment because of its potential environmental and health risks. However, the detection of trace or ultratrace BPA in complicated environmental samples is challenging due to the relatively low affinity and poor selectivity of existing adsorbents used in sample pretreatment. Herein, we report a high-affinity, low environment-dependent and strong interference-resistant abiotic affinity ligand, a N-methacryloyl-l-lysine-NH2 (MLys)-based hydrogel copolymer (HP 17) screened from a small focused polymer library engineered by incorporating various combinations and ratios of candidate functional monomers. The selection of these monomers was guided by molecular mechanism between BPA and the ligand-binding pocket of its estrogen receptors. The BPA–HP17 binding is mainly a synergistic effect of π-cation and hydrophobic interactions. The screened HP 17 has high adsorption capacity (349.4 mg/g) for BPA under wide pH (3.0–10.0) and ionic strength (0–150 mM) range. To improve its practicability, a hydrogel copolymer/Fe3O4 composite microspheres (Fe3O4@HP 17) was synthesized and applied for magnetic solid phase extraction–high-performance liquid chromatography (MSPE–HPLC) analysis of BPA in tap water, lake water and industrial effluents. The method shows wide linear range (2.5⁓100 ng/mL), high sensitivity (detection limit of 0.22 ng/mL even without further concentration after desorption), high accuracies (92.6⁓103.0 %) and good precisions (0.57⁓4.53 %), indicating a great potential of this material and method in the detection of trace or ultratrace BPA in complex environmental water samples.

Unveiling the biological activities of the microbial long chain hydroxy fatty acids as dual agonists of GPR40 and GPR120

The physiological functions of various fatty acid-originating metabolites from foods and fermented products remained mostly untouched. Thereby, this study examined the biological activities of hydroxy fatty acids as agonists of G protein-coupled receptors (i.e., GPR40 and GPR120), which are derived from long-chain fatty acids (e.g., oleic acid and linoleic acid) by microbiota. Cell-based Ca2+ mobilization assays and in silico docking simulations revealed that not only the degree of unsaturation but also the number and position of hydroxyl groups played a key role in their agonist activities. For instance, 8,11-dihydroxyoctadec-9Z-enoic acid exhibited significantly greater Ca2+ response in the GPR40/GPR120-expressing cells as compared to the endogenous agonists (e.g., linoleic acid and docosahexaenoic acid), forming hydrogen bond interactions with residues in the ligand-binding pockets of receptors. This study will contribute to understanding the relationships between fatty acid structures and agonist activities.

A bacterial transcriptionactivator dedicated to the expression of the enzyme catalyzing the first committed step in fatty acid biosynthesis.

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed and rate-limiting step of de novo fatty acid synthesis (FAS). Although this step is tightly regulated, regulators that specifically control transcription of the ACCase genes remain elusive. In this study, we identified LysR-type transcriptional regulator AccR as a dedicated activator for the transcription of accS, a gene encoding a multiple-domain ACCase in Shewanella oneidensis. We showed that AccR interacts with the accS promoter in vivo in response to changes in acetyl-CoA levels and in vitro. Analysis of the crystal structure of the effector-binding domain (EBD) of AccR identified two potential ligand-binding pockets, one of which is likely to bind acetyl-CoA as a ligand based on results from molecular docking, direct binding assay and mutational analysis of the residues predicted to interact with acetyl-CoA. Despite this, the interaction between AccR and acetyl-CoA alone appears unstable, implying that an additional yet unknown ligand is required for activation of AccR. Furthermore, we showed that AccR is acetylated, but the modification may not be critical for sensing acetyl-CoA. Overall, our data substantiate the existence of a dedicated transcriptional regulator for ACCases, expanding our current understanding of the regulation of FAS.

A conserved peptide-binding pocket in HyNaC/ASIC ion channels

The only known peptide-gated ion channels-FaNaCs/WaNaCs and HyNaCs-belong to different clades of the DEG/ENaC family. FaNaCs are activated by the short neuropeptide FMRFamide, and HyNaCs by Hydra RFamides, which are not evolutionarily related to FMRFamide. The FMRFamide-binding site in FaNaCs was recently identified in a cleft atop the large extracellular domain. However, this cleft is not conserved in HyNaCs. Here, we combined molecular modeling and site-directed mutagenesis and identified a putative binding pocket for Hydra-RFamides in the extracellular domain of the heterotrimeric HyNaC2/3/5. This pocket localizes to only one of the three subunit interfaces, indicating that this trimeric ion channel binds a single peptide ligand. We engineered an unnatural amino acid at the putative binding pocket entrance, which allowed covalent tethering of Hydra RFamide to the channel, thereby trapping the channel in an open conformation. The identified pocket localizes to the same region as the acidic pocket of acid-sensing ion channels (ASICs), which binds peptide ligands. The pocket in HyNaCs is less acidic, and both electrostatic and hydrophobic interactions contribute to peptide binding. Collectively, our results reveal a conserved ligand-binding pocket in HyNaCs and ASICs and indicate independent evolution of peptide-binding cavities in the two subgroups of peptide-gated ion channels.

Selective ligand recognition and activation of somatostatin receptors SSTR1 and SSTR3

Somatostatin receptors (SSTRs) exert critical biological functions such as negatively regulating hormone release and cell proliferation, making them popular targets for developing therapeutics to treat endocrine disorders, especially neuroendocrine tumors. Although several panagonists mimicking the endogenous ligand somatostatin are available, the development of more effective and safer somatostatinergic therapies is limited due to a lack of molecular understanding of the ligand recognition and regulation of divergent SSTR subtypes. Here, we report four cryoelectron microscopy structures of Gi-coupled SSTR1 and SSTR3 activated by distinct agonists, including the FDA-approved panagonist pasireotide as well as their selective small molecule agonists L-797591 and L-796778. Our structures reveal a conserved recognition pattern of pasireotide in SSTRs attributed to the binding with a conserved extended binding pocket, distinct from SST14, octreotide, and lanreotide. Together with mutagenesis analyses, our structures further reveal the dynamic feature of ligand binding pockets in SSTR1 and SSTR3 to accommodate divergent agonists, the key determinants of ligand selectivity lying across the orthosteric pocket of different SSTR subtypes, as well as the molecular mechanism underlying diversity and conservation of receptor activation. Our work provides a framework for rational design of subtype-selective SSTR ligands and may facilitate drug development efforts targeting SSTRs with improved therapeutic efficacy and reduced side effects.

The cAMP receptor protein from Gardnerella vaginalis is not regulated by ligands

Overgrowth of Gardnerella vaginalis causes an imbalance in vaginal microecology. The pathogenicity of G. vaginalis is directly regulated by the cAMP receptor protein (CRP). In this study, we resolve the crystal structure of CRPGv at a resolution of 2.22 Å and find some significant differences from homologous proteins. The first 23 amino acids of CRPGv are inserted into the ligand binding pocket, creating a strong steric barrier to ligand entry that has not been seen previously in its homologues. In the absence of ligands, the two α helices used by CRPGv to bind oligonucleotide chains are exposed and can specifically bind TGTGA-N6-TCACA sequences. cAMP and other ligands of CRP homologs are not cofactors of CRPGv. There is no coding gene of the adenylate cyclase, and cAMP could not be identified in G. vaginalis by liquid chromatography tandem mass spectrometry. We speculate that CRPGv may achieve fine regulation through a conformational transformation different from that of its homologous proteins, and this conformational transformation is no longer dependent on small molecules, but may be aided by accessory proteins. CRPGv is the first discovered CRP that is not ligand-regulated, and its active conformation provides a structural basis for drug screening.

Structure-guided discovery of bile acid derivatives for treating liver diseases without causing itch.

Chronic itch is a debilitating symptom profoundly impacting the quality of life in patients with liver diseases like cholestasis. Activation of the human G-protein coupled receptor, MRGPRX4 (hX4), by bile acids (BAs) is implicated in promoting cholestasis itch. However, the detailed underlying mechanisms remain elusive. Here, we identified 3-sulfated BAs that are elevated in cholestatic patients with itch symptoms. We solved the cryo-EM structure of hX4-Gq in a complex with 3-phosphated deoxycholic acid (DCA-3P), a mimic of the endogenous 3-sulfated deoxycholic acid (DCA-3S). This structure revealed an unprecedented ligand-binding pocket in MRGPR family proteins, highlighting the crucial role of the 3-hydroxyl (3-OH) group on BAs in activating hX4. Guided by this structural information, we designed and developed compound 7 (C7), a BA derivative lacking the 3-OH. Notably, C7 effectively alleviates hepatic injury and fibrosis in liver disease models while significantly mitigating the itch side effects.

Discovery of Thiadiazoleamide Derivatives as Potent, Selective, and Orally Available Antagonists Disrupting Androgen Receptor Homodimer.

Androgen receptor (AR) is an important therapeutic target for prostate cancer (PCa) treatment, but prolonged use of AR antagonists has led to variant drug-resistant mutations. Since all marketed AR antagonists target the ligand binding pocket (LBP) of AR, to mitigate cross-resistance, a new drug pocket named Dimer Interface Pocket was discovered and a novel AR antagonist M17-B15 was identified. M17-B15 showed strong in vitro efficacy against PCa but had poor pharmacokinetic properties in vivo. In this study, through rational design and structure-activity relationship exploration, a series of thiadiazoleamide derivatives represented by N29 (IC50 = 0.018 μM) were identified with dominant AR antagonistic activity and remarkable anti-PCa activity in vitro. Furthermore, N29 effectively inhibited a series of typical drug-resistant AR mutants. The improved oral bioavailability of N29 facilitated its efficacy via oral administration, significantly inhibiting LNCaP xenograft tumor in vivo, presenting a promising therapeutic application for PCa.

Molecular Functions and Physiological Roles of Gustatory Receptors of the Silkworm Bombyx mori.

Complete elucidation of members of the gustatory receptor (Gr) family in lepidopteran insects began in the silkworm Bombyx mori. Grs of lepidopteran insects were initially classified into four subfamilies based on the results of phylogenetic studies and analyses of a few ligands. However, with further ligand analysis, it has become clear that plant secondary metabolites are important targets not only for Grs in the bitter subfamily but also for the Drosophila melanogaster Gr43a orthologue subfamily and Grs in the sugar subfamily. Gene knockout experiments showed that B. mori Gr6 (BmGr6) and BmGr9 are involved in the recognition of the feeding-promoting compounds chlorogenic acid and isoquercetin in mulberry leaves by the maxillary palps, suggesting that these Grs are responsible for palpation-dependent host recognition without biting. On the other hand, BmGr expression was also confirmed in nonsensory organs. Midgut enteroendocrine cells that produce specific neuropeptides were shown to express specific BmGrs, suggesting that BmGrs are involved in the induction of endocrine secretion in response to changes in the midgut contents. Furthermore, gene knockout experiments indicated that BmGr6 is indeed involved in the secretion of myosuppressin. On the other hand, BmGr9 was shown to induce signal transduction that is not derived from the intracellular signaling cascade mediated by G proteins but from the fructose-regulated cation channel of BmGr9 itself. Cryogenic electron microscopy revealed the mechanism by which the ion channel of the BmGr9 homotetramer opens upon binding of fructose to the ligand-binding pocket. Research on BmGrs has contributed greatly to our understanding of the functions and roles of Grs in insects.

Integration of Hydrogen-Deuterium Exchange Mass Spectrometry with Molecular Dynamics Simulations and Ensemble Reweighting Enables High Resolution Protein-Ligand Modeling.

Hydrogen-Deuterium exchange mass spectrometry's (HDX-MS) utility in identifying and characterizing protein-small molecule interaction sites has been established. The regions that are seen to be protected from exchange upon ligand binding indicate regions that may be interacting with the ligand, giving a qualitative understanding of the ligand binding pocket. However, quantitatively deriving an accurate high-resolution structure of the protein-ligand complex from the HDX-MS data remains a challenge, often limiting its use in applications such as small molecule drug design. Recent efforts have focused on the development of methods to quantitatively model Hydrogen-Deuterium exchange (HDX) data from computationally modeled structures to garner atomic level insights from peptide-level resolution HDX-MS. One such method, HDX ensemble reweighting (HDXer), employs maximum entropy reweighting of simulated HDX data to experimental HDX-MS to model structural ensembles. In this study, we implement and validate a workflow which quantitatively leverages HDX-MS data to accurately model protein-small molecule ligand interactions. To that end, we employ a strategy combining computational protein-ligand docking, molecular dynamics simulations, HDXer, and dimensional reduction and clustering approaches to extract high-resolution drug binding poses that most accurately conform with HDX-MS data. We apply this workflow to model the interaction of ERK2 and FosA with small molecule compounds and inhibitors they are known to bind. In five out of six of the protein-ligand pairs tested, the HDX derived protein-ligand complexes result in a ligand root-mean-square deviation (RMSD) within 2.5 Å of the known crystal structure ligand.

Carnosic acid, a novel food-source AT1R antagonist and its anti-hypertension mechanism

Hypertension is the most prevalent non-communicable disease, affecting billions of people worldwide. Discovery and development of natural antihypertensive lead compounds or drugs are important to resolve the limitations of existing antihypertensive drug safety and resistance. This investigation verified that carnosic acid (CA), an important active ingredient of rosemary, an edible spice plant, indicates a significant anti-hypertensive activity in spontaneous hypertension rats by targeting AT1R. Moreover, our research indicated that CA shared a comparable antagonistic mechanism with established synthetic angiotensin II receptor blockers (ARBs), as it occupies the binding sites of Angiotensin II (AngII) at His6 and Pro7 within the AT1R's ligand-binding pocket. Notably, CA exerted better anti-hypertensive activity since it could not break the Asn1113.35-Asn2957.46 hydrogen bond to stabilize the AT1R inactive state. As the first potent AT1R antagonist identified in a natural food source, CA is poised to become a novel anti-hypertensive lead compound, distinguished by its unique skeleton structure different from conventional ARBs. This research lays a valuable theoretical groundwork for the future exploration of CA and rosemary extract in both fundamental studies and clinical applications.

Targeting STING oligomerization with licochalcone D ameliorates STING-driven inflammatory diseases.

The development of STING inhibitors for the treatment of STING-related inflammatory diseases continues to encounter significant challenges. The activation of STING is a multi-step process that includes binding with cGAMP, self-oligomerization, and translocation from the endoplasmic reticulum to the Golgi apparatus, ultimately inducing the expression of IRF3 and NF-κB-mediated interferons and inflammatory cytokines. It has been demonstrated that disruption of any of these steps can effectively inhibit STING activation. Traditional structure-based drug screening methodologies generally focus on specific binding sites. In this study, a TransformerCPI model based on protein primary sequences and independent of binding sites is employed to identify compounds capable of binding to the STING protein. The natural product Licochalcone D (LicoD) is identified as a potent and selective STING inhibitor. LicoD does not bind to the classical ligand-binding pocket; instead, it covalently modifies the Cys148 residue of STING. This modification inhibits STING oligomerization, consequently suppressing the recruitment of TBK1 and the nuclear translocation of IRF3 and NF-κB. LicoD treatment ameliorates the inflammatory phenotype in Trex1-1- mice and inhibits the progression of DSS-induced colitis and AOM/DSS-induced colitis-associated colon cancer (CAC). In summary, this study reveals the potential of LicoD in treating STING-driven inflammatory diseases. It also demonstrates the utility of the TransformerCPI model in discovering allosteric compounds beyond the conventional binding pockets.

Structure and Function of Somatostatin and its Receptors in Endocrinology.

Somatostatin analogs, such as octreotide (OCT), lanreotide, and pasireotide, which function as somatostatin receptor ligands (SRLs), are the main drugs used for the treatment of acromegaly. These ligands are also used as important molecules for radiation therapy and imaging of neuroendocrine tumors (NETs). Somatostatin receptors (SSTRs) are canonical G protein-coupled proteins (GPCRs) that play a role in metabolism, growth, and pathological conditions such as hormone disorders, neurological diseases, and cancers. Cryogenic electron microscopy (cryo-EM) combined with the protein structure prediction platform AlphaFold has been used to determine the three-dimensional structures of many proteins. Recently, several groups published a series of papers illustrating the three-dimensional structure of SSTR2, including that of the inactive/activated SSTR2-G protein complex bound to different ligands. The results revealed the residues that contribute to the ligand binding pocket and demonstrated that Trp8-Lys9 (the W-K motif) in somatostatin analogs is the key motif in stabilizing the bottom part of the binding pocket. In this review, we discuss the recent findings related to the structural analysis of SSTRs and SRLs, the relationships between the structural data and clinical findings, and the future development of novel structure-based therapies.

Decoration of GW9662 scaffold with a fragment-like hit via copper-catalyzed azide-alkyne cycloaddition reaction into peroxisome proliferator-activated receptor γ agonists

PPARγ is a member of nuclear receptor superfamily, which possesses a large Y-shaped cavity in the ligand-binding pocket. GW9662 is a well-known PPARγ antagonist which covalently reacts with the Cys285 residue via nucleophilic substitution. In contrast to its irreversibility, the ability of GW9662 to prevent other ligands from accessing the ligand-binding pocket is partly defective. By focusing on this incompleteness, we develop an integrated approach to create a new PPARγ agonist by using a structure of GW9662 as a scaffold for linking with a fragment compound. A screening of 1,040 compounds identified a partner ligand which synergistically activates PPARγ transcription in combination with GW9662. We introduced an azido or alkyne group to GW9662 or the identified fragment hit, respectively, and then connected the two structures via copper-catalyzed azide-alkyne cycloaddition. A coupling reaction provided a series of hybrid structure with triazole linker, among which the compounds with a bent configuration function as a partial PPARγ agonist. These results highlight a potential utility of GW9662 for the decoration with a fragment compound to develop a covalent agonist for PPARγ activation.

Abstract Tu149: FDA-Approved Antidepressant Trazodone Potentially Increases the Risk of Dyslipidemia

Cardiovascular disease is the leading cause of death. Many cardiovascular health problems, such as atherosclerosis, are caused by dyslipidemia. One xenobiotic nuclear receptor, Pregnane X Receptor (PXR), plays a significant role in atherosclerosis and dyslipidemia, and is activated by various environmental chemicals, including endocrine-disrupting chemicals (EDCs). EDCs are found in common household items such as plastics, medications, and food. Trazodone is a clinically used medication to treat depression by aiding in restoring the balance of serotonin in the brain. But it is unclear if Trazodone has possible impacts on cardiovascular risk factors such as dyslipidemia. Our preliminary data suggested that Trazodone activated human PXR in both intestinal (LS180) and hepatic (HepG2) cells. We hypothesize that Trazodone could regulate the cholesterol uptake mediated by PXR signaling. In this study we use cell-based transfection assay to evaluate the underlying mechanisms by which Trazodone activates PXR. We found that Trazodone was a more potent agonist of human PXR than mouse PXR. Trazodone could activate PXR more intensely in human liver cells compared with human intestinal cells. Our data suggested that Trazodone was a selective PXR agonist and promoted the dissociation between PXR and its nuclear corepressors. Next, we are to identify the key amino acid residues within PXR ligand binding pocket that interact with Trazodone by using computational docking study along with site-mutagenesis assay. Furthermore, we plan to estimate if Trazodone altered cholesterol uptake by human intestinal cells using fluorescence-labeled cholesterol. In conclusion, we explore the potential molecular mechanisms of how FDA-approved antidepressant Trazodone activates human PXR and increases the possible risk of dyslipidemia, which provides potential evidence on future cardiovascular disease risk assessment for Trazodone as well as other antidepressant drugs.

Structure-based insights into fluorogenic RNA aptamers.

Fluorogenic RNA aptamers are in vitro-selected RNA molecules capable of binding to specific fluorophores, significantly increasing their intrinsic fluorescence. Over the past decade, the color palette of fluorescent RNA aptamers has greatly expanded. The emergence and development of these fluorogenic RNA aptamers has introduced a powerful approach for visualizing RNA localization and transport with high spatiotemporal resolution in live cells. To date, a variety of tertiary structures of fluorogenic RNA aptamers have been determined using X-ray crystallography or NMR spectroscopy. Many of these fluorogenic RNA aptamers feature base quadruples or base triples in their fluorophore-binding sites. This review summarizes the structure-based investigations of fluorogenic RNA aptamers, with a focus on their overall folds, ligand-binding pockets and fluorescence activation mechanisms. Additionally, the exploration of how structures guide rational optimization to enhance RNA visualization techniques is discussed.

Structural basis for the full and partial agonist activities of retinoid X receptor α ligands with an iso-butoxy and an isopropyl group

Retinoid X receptors (RXRs) belong to a retinoid-binding subgroup of the nuclear receptor family, and their synthetic agonists have been developed as therapeutics for glucose and lipid metabolism, inflammation, and inflammatory bowel disease, although RXR agonists could cause side effects such as hypothyroidism, hypertriglyceridemia, and hepatomegaly. We previously reported novel full and partial agonists, NEt–3IB and NEt–4IB, which reduce the side effects, but the molecular basis of their different activity was not clear. In this study, we report the crystal structures of the ligand-binding domain of human RXRα complexed with NEt–3IB and NEt–4IB. Detailed comparisons of the two structures showed that the full agonist, NEt–3IB, is more stably accommodated in the ligand-binding pocket due to the interactions of the bulky iso-butoxy group with helices 5 and 7. The stabilization of these helices led to the stabilization of helix 12, which is important for formation of the coactivator-binding site. The structures shed light on the novel mechanism of the regulation of RXR activity through the interaction between the bound agonist and helix 7, an interaction that was not previously considered important.

Human P2X4 receptor gating is modulated by a stable cytoplasmic cap and a unique allosteric pocket.

P2X receptors (P2XRs) are a family of ATP-gated ion channels comprising homomeric and heteromeric trimers of seven subunits (P2X 1 - P2X 7 ) that confer different rates of desensitization. The helical recoil model of P2XR desensitization proposes the stability of the cytoplasmic cap sets the rate of desensitization, but timing of its formation is unclear for slow-desensitizing P2XRs. We report cryo-EM structures of full-length, wild-type human P2X 4 receptor in apo, antagonist-bound, and desensitized states. Because the apo and antagonist-bound structures of this slow-desensitizing P2XR include an intact cytoplasmic cap while the desensitized state structure does not, the cytoplasmic cap forms before agonist binding. Furthermore, structural and functional data suggests the cytoplasmic cap is stabilized by lipids to slow desensitization and that P2X 4 is further modified by glycosylation and palmitoylation. Finally, our antagonist-bound inhibited state structure reveals features specific to the allosteric ligand-binding pocket in human receptors that empower the development of small-molecule modulators.