RÉSUMÉ
Bombesin receptor subtype-3 (BRS3) is an important orphan G protein-coupled receptor that regulates energy homeostasis and insulin secretion. As a member of the bombesin receptor (BnR) family, the lack of known endogenous ligands and high-resolution structure has hindered the understanding of BRS3 signaling and function. We present two cryogenic electron microscopy (cryo-EM) structures of BRS3 in complex with the heterotrimeric Gq protein in its active states: one bound to the pan-BnR agonist BA1 and the other bound to the synthetic BRS3-specific agonist MK-5046. These structures reveal the architecture of the orthosteric ligand pocket underpinning molecular recognition and provide insights into the structural basis for BRS3's selectivity and low affinity for bombesin peptides. Examination of conserved micro-switches suggests a shared activation mechanism among BnRs. Our findings shed light on BRS3's ligand selectivity and signaling mechanisms, paving the way for exploring its therapeutic potential for diabetes, obesity, and related metabolic disorders.
RÉSUMÉ
Kisspeptin signaling through its G protein-coupled receptor, KISS1R, plays an indispensable role in regulating reproduction via the hypothalamic-pituitary-gonadal axis. Dysregulation of this pathway underlies severe disorders like infertility and precocious puberty. Here, we present cryo-EM structures of KISS1R bound to the endogenous agonist kisspeptin-10 and a synthetic analog TAK-448. These structures reveal pivotal interactions between peptide ligands and KISS1R extracellular loops for receptor activation. Both peptides exhibit a conserved binding mode, unveiling their common activation mechanism. Intriguingly, KISS1R displays a distinct 40° angular deviation in its intracellular TM6 region compared to other Gq-coupled receptors, enabling distinct interactions with Gq. This study reveals the molecular intricacies governing ligand binding and activation of KISS1R, while highlighting its exceptional ability to couple with Gq. Our findings pave the way for structure-guided design of therapeutics targeting this physiologically indispensable receptor.
Sujet(s)
Sous-unités alpha Gq-G11 des protéines G , Kisspeptines , Récepteur de la Kisspeptine-1 , Humains , Récepteur de la Kisspeptine-1/métabolisme , Kisspeptines/métabolisme , Kisspeptines/composition chimique , Sous-unités alpha Gq-G11 des protéines G/métabolisme , Sous-unités alpha Gq-G11 des protéines G/composition chimique , Liaison aux protéines , Cellules HEK293 , Cryomicroscopie électroniqueRÉSUMÉ
Platelet-activating factor (PAF) is a potent phospholipid mediator crucial in multiple inflammatory and immune responses through binding and activating the PAF receptor (PAFR). However, drug development targeting the PAFR has been limited, partly due to an incomplete understanding of its activation mechanism. Here, we present a 2.9-Å structure of the PAF-bound PAFR-Gi complex. Structural and mutagenesis analyses unveil a specific binding mode of PAF, with the choline head forming cation-π interactions within PAFR hydrophobic pocket, while the alkyl tail penetrates deeply into an aromatic cleft between TM4 and TM5. Binding of PAF modulates conformational changes in key motifs of PAFR, triggering the outward movement of TM6, TM7, and helix 8 for G protein coupling. Molecular dynamics simulation suggests a membrane-side pathway for PAF entry into PAFR via the TM4-TM5 cavity. By providing molecular insights into PAFR signaling, this work contributes a foundation for developing therapeutic interventions targeting PAF signal axis.
Sujet(s)
Facteur d'activation plaquettaire , Glycoprotéines de membrane plaquettaire , Récepteurs couplés aux protéines G , Glycoprotéines de membrane plaquettaire/métabolisme , Glycoprotéines de membrane plaquettaire/composition chimique , Facteur d'activation plaquettaire/métabolisme , Récepteurs couplés aux protéines G/métabolisme , Récepteurs couplés aux protéines G/composition chimique , Humains , Simulation de dynamique moléculaire , Liaison aux protéines , Sites de fixation , Cellules HEK293 , Transduction du signalRÉSUMÉ
As the most abundant organic substances in nature, carbohydrates are essential for life. Understanding how carbohydrates regulate proteins in the physiological and pathological processes presents opportunities to address crucial biological problems and develop new therapeutics. However, the diversity and complexity of carbohydrates pose a challenge in experimentally identifying the sites where carbohydrates bind to and act on proteins. Here, we introduce a deep learning model, DeepGlycanSite, capable of accurately predicting carbohydrate-binding sites on a given protein structure. Incorporating geometric and evolutionary features of proteins into a deep equivariant graph neural network with the transformer architecture, DeepGlycanSite remarkably outperforms previous state-of-the-art methods and effectively predicts binding sites for diverse carbohydrates. Integrating with a mutagenesis study, DeepGlycanSite reveals the guanosine-5'-diphosphate-sugar-recognition site of an important G-protein coupled receptor. These findings demonstrate DeepGlycanSite is invaluable for carbohydrate-binding site prediction and could provide insights into molecular mechanisms underlying carbohydrate-regulation of therapeutically important proteins.
Sujet(s)
Apprentissage profond , Sites de fixation , Glucides/composition chimique , Liaison aux protéines , , Humains , Protéines/métabolisme , Protéines/composition chimique , Modèles moléculairesRÉSUMÉ
The neuropeptide 26RFa, a member of the RF-amide peptide family, activates the pyroglutamylated RF-amide peptide receptor (QRFPR), a class A GPCR. The 26RFa/QRFPR system plays critical roles in energy homeostasis, making QRFPR an attractive drug target for treating obesity, diabetes, and eating disorders. However, the lack of structural information has hindered our understanding of the peptide recognition and regulatory mechanism of QRFPR, impeding drug design efforts. In this study, we determined the cryo-EM structure of the Gq-coupled QRFPR bound to 26RFa. The structure reveals a unique assembly mode of the extracellular region of the receptor and the N-terminus of the peptide, and elucidates the recognition mechanism of the C-terminal heptapeptide of 26RFa by the transmembrane binding pocket of QRFPR. The study also clarifies the similarities and distinctions in the binding pattern of the RF-amide moiety in five RF-amide peptides and the RY-amide segment in neuropeptide Y. These findings deepen our understanding of the RF-amide peptide recognition, aiding in the rational design of drugs targeting QRFPR and other RF-amide peptide receptors.
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The noradrenaline transporter has a pivotal role in regulating neurotransmitter balance and is crucial for normal physiology and neurobiology1. Dysfunction of noradrenaline transporter has been implicated in numerous neuropsychiatric diseases, including depression and attention deficit hyperactivity disorder2. Here we report cryo-electron microscopy structures of noradrenaline transporter in apo and substrate-bound forms, and as complexes with six antidepressants. The structures reveal a noradrenaline transporter dimer interface that is mediated predominantly by cholesterol and lipid molecules. The substrate noradrenaline binds deep in the central binding pocket, and its amine group interacts with a conserved aspartate residue. Our structures also provide insight into antidepressant recognition and monoamine transporter selectivity. Together, these findings advance our understanding of noradrenaline transporter regulation and inhibition, and provide templates for designing improved antidepressants to treat neuropsychiatric disorders.
Sujet(s)
Antidépresseurs , Cryomicroscopie électronique , Transporteurs de la norépinéphrine , Norépinéphrine , Multimérisation de protéines , Humains , Antidépresseurs/composition chimique , Antidépresseurs/métabolisme , Antidépresseurs/pharmacologie , Apoprotéines/composition chimique , Apoprotéines/métabolisme , Apoprotéines/ultrastructure , Acide aspartique/composition chimique , Acide aspartique/métabolisme , Sites de fixation , Cholestérol/métabolisme , Cholestérol/composition chimique , Modèles moléculaires , Norépinéphrine/métabolisme , Norépinéphrine/composition chimique , Transporteurs de la norépinéphrine/antagonistes et inhibiteurs , Transporteurs de la norépinéphrine/composition chimique , Transporteurs de la norépinéphrine/métabolisme , Transporteurs de la norépinéphrine/ultrastructure , Liaison aux protéines , Spécificité du substratRÉSUMÉ
Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that regulates food intake, energy balance, and other physiological functions by stimulating MCHR1 and MCHR2 receptors, both of which are class A G protein-coupled receptors. MCHR1 predominately couples to inhibitory G protein, Gi/o, and MCHR2 can only couple to Gq/11. Here we present cryo-electron microscopy structures of MCH-activated MCHR1 with Gi and MCH-activated MCHR2 with Gq at the global resolutions of 3.01 Å and 2.40 Å, respectively. These structures reveal that MCH adopts a consistent cysteine-mediated hairpin loop configuration when bound to both receptors. A central arginine from the LGRVY core motif between the two cysteines of MCH penetrates deeply into the transmembrane pocket, triggering receptor activation. Integrated with mutational and functional insights, our findings elucidate the molecular underpinnings of ligand recognition and MCH receptor activation and offer a structural foundation for targeted drug design.
RÉSUMÉ
G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, with many GPCRs having crucial roles in endocrinology and metabolism. Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly regarding GPCRs, over the past decade. Since the first pair of GPCR structures resolved by cryo-EM were published in 2017, the number of GPCR structures resolved by cryo-EM has surpassed the number resolved by X-ray crystallography by 30%, reaching >650, and the number has doubled every ~0.63 years for the past 6 years. At this pace, it is predicted that the structure of 90% of all human GPCRs will be completed within the next 5-7 years. This Review highlights the general structural features and principles that guide GPCR ligand recognition, receptor activation, G protein coupling, arrestin recruitment and regulation by GPCR kinases. The Review also highlights the diversity of GPCR allosteric binding sites and how allosteric ligands could dictate biased signalling that is selective for a G protein pathway or an arrestin pathway. Finally, the authors use the examples of glycoprotein hormone receptors and glucagon-like peptide 1 receptor to illustrate the effect of cryo-EM on understanding GPCR biology in endocrinology and metabolism, as well as on GPCR-related endocrine diseases and drug discovery.
Sujet(s)
Cryomicroscopie électronique , Découverte de médicament , Récepteurs couplés aux protéines G , Cryomicroscopie électronique/méthodes , Humains , Récepteurs couplés aux protéines G/métabolisme , Récepteurs couplés aux protéines G/composition chimique , Découverte de médicament/méthodes , Endocrinologie/méthodes , Animaux , Transduction du signal , LigandsRÉSUMÉ
Geometric deep learning has been revolutionizing the molecular modeling field. Despite the state-of-the-art neural network models are approaching ab initio accuracy for molecular property prediction, their applications, such as drug discovery and molecular dynamics (MD) simulation, have been hindered by insufficient utilization of geometric information and high computational costs. Here we propose an equivariant geometry-enhanced graph neural network called ViSNet, which elegantly extracts geometric features and efficiently models molecular structures with low computational costs. Our proposed ViSNet outperforms state-of-the-art approaches on multiple MD benchmarks, including MD17, revised MD17 and MD22, and achieves excellent chemical property prediction on QM9 and Molecule3D datasets. Furthermore, through a series of simulations and case studies, ViSNet can efficiently explore the conformational space and provide reasonable interpretability to map geometric representations to molecular structures.
RÉSUMÉ
Allosteric regulation, induced by perturbations at an allosteric site topographically distinct from the orthosteric site, is one of the most direct and efficient ways to fine-tune macromolecular function. The Allosteric Database (ASD; accessible online at http://mdl.shsmu.edu.cn/ASD) has been systematically developed since 2009 to provide comprehensive information on allosteric regulation. In recent years, allostery has seen sustained growth and wide-ranging applications in life sciences, from basic research to new therapeutics development, while also elucidating emerging obstacles across allosteric research stages. To overcome these challenges and maintain high-quality data center services, novel features were curated in the ASD2023 update: (i) 66 589 potential allosteric sites, covering > 80% of the human proteome and constituting the human allosteric pocketome; (ii) 748 allosteric protein-protein interaction (PPI) modulators with clear mechanisms, aiding protein machine studies and PPI-targeted drug discovery; (iii) 'Allosteric Hit-to-Lead,' a pioneering dataset providing panoramic views from 87 well-defined allosteric hits to 6565 leads and (iv) 456 dualsteric modulators for exploring the simultaneous regulation of allosteric and orthosteric sites. Meanwhile, ASD2023 maintains a significant growth of foundational allosteric data. Based on these efforts, the allosteric knowledgebase is progressively evolving towards an integrated landscape, facilitating advancements in allosteric target identification, mechanistic exploration and drug discovery.
Sujet(s)
Site allostérique , Bases de connaissances , Humains , Régulation allostérique , Découverte de médicament , Ligands , Protéome , Cartes d'interactions protéiquesRÉSUMÉ
Trace amine-associated receptor 1 (TAAR1), the founding member of a nine-member family of trace amine receptors, is responsible for recognizing a range of biogenic amines in the brain, including the endogenous ß-phenylethylamine (ß-PEA)1 as well as methamphetamine2, an abused substance that has posed a severe threat to human health and society3. Given its unique physiological role in the brain, TAAR1 is also an emerging target for a range of neurological disorders including schizophrenia, depression and drug addiction2,4,5. Here we report structures of human TAAR1-G-protein complexes bound to methamphetamine and ß-PEA as well as complexes bound to RO5256390, a TAAR1-selective agonist, and SEP-363856, a clinical-stage dual agonist for TAAR1 and serotonin receptor 5-HT1AR (refs. 6,7). Together with systematic mutagenesis and functional studies, the structures reveal the molecular basis of methamphetamine recognition and underlying mechanisms of ligand selectivity and polypharmacology between TAAR1 and other monoamine receptors. We identify a lid-like extracellular loop 2 helix/loop structure and a hydrogen-bonding network in the ligand-binding pockets, which may contribute to the ligand recognition in TAAR1. These findings shed light on the ligand recognition mode and activation mechanism for TAAR1 and should guide the development of next-generation therapeutics for drug addiction and various neurological disorders.
Sujet(s)
Métamfétamine , Phénéthylamines , Récepteurs couplés aux protéines G , Humains , Ligands , Métamfétamine/métabolisme , Maladies du système nerveux/métabolisme , Phénéthylamines/métabolisme , Récepteurs couplés aux protéines G/agonistes , Récepteurs couplés aux protéines G/composition chimique , Récepteurs couplés aux protéines G/métabolisme , Troubles liés à une substance/métabolisme , Protéines G hétérotrimériques/métabolisme , Polypharmacologie , Liaison hydrogèneRÉSUMÉ
As a major class of biomolecules, carbohydrates play indispensable roles in various biological processes. However, it remains largely unknown how carbohydrates directly modulate important drug targets, such as G-protein coupled receptors (GPCRs). Here, we employed P2Y purinoceptor 14 (P2Y14), a drug target for inflammation and immune responses, to uncover the sugar nucleotide activation of GPCRs. Integrating molecular dynamics simulation with functional study, we identified the uridine diphosphate (UDP)-sugar-binding site on P2Y14, and revealed that a UDP-glucose might activate the receptor by bridging the transmembrane (TM) helices 2 and 7. Between TM2 and TM7 of P2Y14, a conserved salt bridging chain (K2.60-D2.64-K7.35-E7.36 [KDKE]) was identified to distinguish different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine. We identified the KDKE chain as a conserved functional motif of sugar binding for both P2Y14 and P2Y purinoceptor 12 (P2Y12), and then designed three sugar nucleotides as agonists of P2Y12. These results not only expand our understanding for activation of purinergic receptors but also provide insights for the carbohydrate drug development for GPCRs.
Sugars and other types of carbohydrates are biomolecules which play a range of key roles in the body. In particular, they are important messengers that help to coordinate immune responses. For example, a carbohydrate known as UDP-Glucose (a kind of UDP-sugar) can activate P2Y14, a receptor studded through the surface of many cells; this event then triggers a cascade of molecular events associated with asthma, kidney injury and lung inflammation. Yet it remains unclear how exactly UDP-Glucose recognizes P2Y14 and, more broadly, how carbohydrates interact with purinergic receptors, the class of proteins that P2Y14 belongs to. To examine this question, Zhao et al. combined functional experiments in the laboratory with molecular dynamics simulations, a computational approach. This work revealed that UDP-Glucose may activate P2Y14 by bridging its segments anchored within the cell membrane. A component of P2Y14, known as the KDKE chain, was found to have an important role in distinguishing between highly similar types of UDP-sugars. This allowed Zhao et al. to design three sugar molecules which could activate another purinergic receptor that also contained a KDKE chain. Purinergic receptors are promising therapeutic targets. A finer understanding of how they recognise the molecules that activate them is therefore important to be able to identify and design new drug compounds.
Sujet(s)
Nucléotides , Récepteurs purinergiques , Uridine diphosphate glucose , Sucres , Récepteurs purinergiques P2YRÉSUMÉ
Niacin, an age-old lipid-lowering drug, acts through the hydroxycarboxylic acid receptor 2 (HCAR2), a G-protein-coupled receptor (GPCR). Yet, its use is hindered by side effects like skin flushing. To address this, specific HCAR2 agonists, like MK-6892 and GSK256073, with fewer adverse effects have been created. However, the activation mechanism of HCAR2 by niacin and these new agonists is not well understood. Here, we present three cryoelectron microscopy structures of Gi-coupled HCAR2 bound to niacin, MK-6892, and GSK256073. Our findings show that different ligands induce varying binding pockets in HCAR2, influenced by aromatic amino acid clusters (W91ECL1, H1614.59, W1885.38, H1895.39, and F1935.43) from receptors ECL1, TM4, and TM5. Additionally, conserved residues R1113.36 and Y2847.43, unique to the HCA receptor family, likely initiate activation signal propagation in HCAR2. This study provides insights into ligand recognition, receptor activation, and G protein coupling mediated by HCAR2, laying the groundwork for developing HCAR2-targeted drugs.
Sujet(s)
Acides cyclohexanecarboxyliques , Acide nicotinique , Humains , Acide nicotinique/pharmacologie , Cryomicroscopie électronique , Récepteurs couplés aux protéines G/métabolisme , Ligands , LipidesRÉSUMÉ
The G protein-coupled receptor 35 (GPR35) has been identified as a potential target in the treatment of inflammatory bowel disease (IBD). However, the lack of high and equipotent agonists on both human and mouse GPR35 has limited the in vivo study of GPR35 agonists in mouse models of IBD. In this study, structural modifications to lodoxamide provides a series of high and equivalent agonists on human, mouse, and rat GPR35. These molecules eliminate the species selectivity of human to mouse and rat orthologs that have been prevalent with GPR35 agonists including lodoxamide. The cLogP properties are also optimized to make the compounds more obedient to drug-like rules, yielding compound 4b (cLogP = 2.41), which activates human, mouse or rat GPR35 with EC50 values of 76.0, 63.7 and 77.8 nM, respectively. Oral administration of compound 4b at 20 mg/kg alleviates clinical symptoms of DSS-induced IBD in mice, and is slightly more effective than 5-ASA at 200 mg/kg. In summary, it can serve as a new start point for exploiting more potent GPR35 agonists without species differences for the treatment of IBD, and warrants further study.
Sujet(s)
Maladies inflammatoires intestinales , Récepteurs couplés aux protéines G , Rats , Souris , Humains , Animaux , Récepteurs couplés aux protéines G/agonistes , Acide oxamique/pharmacologie , Maladies inflammatoires intestinales/traitement médicamenteux , Administration par voie oraleRÉSUMÉ
Chiral recognition of amino acid plays a significant role in pharmaceutical, medical, and food science. This study describes a chiral sensing system of ß-cyclodextrin (ß-CD)-coated sulfur quantum dots (CD-SQDs) for the selective fluorescence recognition of tryptophan (Trp) enantiomers. CD-SQDs were prepared by a facile assembly fission method and could selectively recognize L-Trp by the different binding ability between L/D-Trp and ß-CD. The inclusion of L-Trp and the stereoselective catalysis of CD-SQDs enzyme mimics cause the increased fluorescence intensity of CD-SQDs, which has a linear response ranging from 10 to 500 nM and the detection limit as 2.3 nM. CD-SQDs also show great selectivity for L-Trp from the commercial compound amino acid injection. The study could provide an effective method for the chiral recognition of amino acid enantiomers based on the catalytic activity of nanoenzymes.
Sujet(s)
Boîtes quantiques , Cyclodextrines bêta , Tryptophane , Boîtes quantiques/composition chimique , Cyclodextrines bêta/composition chimique , StéréoisomérieRÉSUMÉ
The stimulator of interferon genes (STING) is an important therapeutic target for cancer diseases. The activated STING recruits downstream tank-binding kinase 1 (TBK1) to trigger several important immune responses. However, the molecular mechanism of how agonist molecules mediate the STING-TBK1 interactions remains elusive. Here, we performed molecular dynamics simulations to capture the conformational changes of STING and TBK1 upon agonist binding. Our simulations revealed that multiple helices (α5-α7) and especially three loops (loop 6, loop 8, and C-terminal tail) of STING participated in the allosteric mediation of the STING-TBK1 interactions. Consistent results were also observed in the simulations of the constitutive activating mutant of STING (R284S). We further identified α5 as a key region in this agonist-induced activation mechanism of STING. Free-energy perturbation calculations of multiple STING agonists demonstrated that an alkynyl group targeting α5 is a determinant for agonist activities. These results not only offer deeper insights into the agonist-induced allosteric mediation of STING-TKB1 interactions but also provide a guidance for future drug development of this important therapeutic target.