RESUMEN
Opioids are effective analgesics, but their use is beset by serious side effects, including addiction and respiratory depression, which contribute to the ongoing opioid crisis. The human opioid system contains four opioid receptors (µOR, δOR, κOR, and NOPR) and a set of related endogenous opioid peptides (EOPs), which show distinct selectivity toward their respective opioid receptors (ORs). Despite being key to the development of safer analgesics, the mechanisms of molecular recognition and selectivity of EOPs to ORs remain unclear. Here, we systematically characterize the binding of EOPs to ORs and present five structures of EOP-OR-Gi complexes, including ß-endorphin- and endomorphin-bound µOR, deltorphin-bound δOR, dynorphin-bound κOR, and nociceptin-bound NOPR. These structures, supported by biochemical results, uncover the specific recognition and selectivity of opioid peptides and the conserved mechanism of opioid receptor activation. These results provide a structural framework to facilitate rational design of safer opioid drugs for pain relief.
Asunto(s)
Receptores Opioides , Humanos , Analgésicos Opioides/farmacología , Péptidos Opioides , Receptores Opioides mu/metabolismo , Receptores Opioides/químicaRESUMEN
Morphine and fentanyl are among the most used opioid drugs that confer analgesia and unwanted side effects through both G protein and arrestin signaling pathways of µ-opioid receptor (µOR). Here, we report structures of the human µOR-G protein complexes bound to morphine and fentanyl, which uncover key differences in how they bind the receptor. We also report structures of µOR bound to TRV130, PZM21, and SR17018, which reveal preferential interactions of these agonists with TM3 side of the ligand-binding pocket rather than TM6/7 side. In contrast, morphine and fentanyl form dual interactions with both TM3 and TM6/7 regions. Mutations at the TM6/7 interface abolish arrestin recruitment of µOR promoted by morphine and fentanyl. Ligands designed to reduce TM6/7 interactions display preferential G protein signaling. Our results provide crucial insights into fentanyl recognition and signaling of µOR, which may facilitate rational design of next-generation analgesics.
Asunto(s)
Fentanilo , Morfina , Humanos , Analgésicos Opioides/farmacología , Arrestina/metabolismo , Fentanilo/farmacología , Proteínas de Unión al GTP/metabolismo , Morfina/farmacología , Receptores Opioides muRESUMEN
The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.
Asunto(s)
Receptores de Dopamina D1/química , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Transducción de Señal , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/análogos & derivados , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Secuencia de Aminoácidos , Secuencia Conservada , Microscopía por Crioelectrón , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Dopamina D1/ultraestructura , Receptores de Dopamina D2/ultraestructura , Homología Estructural de ProteínaRESUMEN
The metabolic challenges present in tumors attenuate the metabolic fitness and antitumor activity of tumor-infiltrating T lymphocytes (TILs). However, it remains unclear whether persistent metabolic insufficiency can imprint permanent T cell dysfunction. We found that TILs accumulated depolarized mitochondria as a result of decreased mitophagy activity and displayed functional, transcriptomic and epigenetic characteristics of terminally exhausted T cells. Mechanistically, reduced mitochondrial fitness in TILs was induced by the coordination of T cell receptor stimulation, microenvironmental stressors and PD-1 signaling. Enforced accumulation of depolarized mitochondria with pharmacological inhibitors induced epigenetic reprogramming toward terminal exhaustion, indicating that mitochondrial deregulation caused T cell exhaustion. Furthermore, supplementation with nicotinamide riboside enhanced T cell mitochondrial fitness and improved responsiveness to anti-PD-1 treatment. Together, our results reveal insights into how mitochondrial dynamics and quality orchestrate T cell antitumor responses and commitment to the exhaustion program.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Recuento de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Dinámicas Mitocondriales/inmunología , Biomarcadores , Epigénesis Genética , Epigenómica , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/inmunología , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Mitofagia , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/terapia , Niacinamida/farmacología , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Estrés Fisiológico , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Naturally occurring (native) sugars and carbohydrates contain numerous hydroxyl groups of similar reactivity1,2. Chemists, therefore, rely typically on laborious, multi-step protecting-group strategies3 to convert these renewable feedstocks into reagents (glycosyl donors) to make glycans. The direct transformation of native sugars to complex saccharides remains a notable challenge. Here we describe a photoinduced approach to achieve site- and stereoselective chemical glycosylation from widely available native sugar building blocks, which through homolytic (one-electron) chemistry bypasses unnecessary hydroxyl group masking and manipulation. This process is reminiscent of nature in its regiocontrolled generation of a transient glycosyl donor, followed by radical-based cross-coupling with electrophiles on activation with light. Through selective anomeric functionalization of mono- and oligosaccharides, this protecting-group-free 'cap and glycosylate' approach offers straightforward access to a wide array of metabolically robust glycosyl compounds. Owing to its biocompatibility, the method was extended to the direct post-translational glycosylation of proteins.
Asunto(s)
Técnicas de Química Sintética , Oligosacáridos , Azúcares , Radicales Libres/química , Radicales Libres/metabolismo , Glicosilación/efectos de la radiación , Indicadores y Reactivos/química , Luz , Oligosacáridos/síntesis química , Oligosacáridos/química , Oligosacáridos/metabolismo , Oligosacáridos/efectos de la radiación , Estereoisomerismo , Azúcares/síntesis química , Azúcares/química , Azúcares/metabolismo , Azúcares/efectos de la radiaciónRESUMEN
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.
Asunto(s)
Antidepresivos , Microscopía por Crioelectrón , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática , Norepinefrina , Multimerización de Proteína , Humanos , Antidepresivos/química , Antidepresivos/metabolismo , Antidepresivos/farmacología , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Sitios de Unión , Colesterol/metabolismo , Colesterol/química , Modelos Moleculares , Norepinefrina/metabolismo , Norepinefrina/química , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/química , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/ultraestructura , Unión Proteica , Especificidad por SustratoRESUMEN
Calcium-oxygen (Ca-O2) batteries can theoretically afford high capacity by the reduction of O2 to calcium oxide compounds (CaOx) at low cost1-5. Yet, a rechargeable Ca-O2 battery that operates at room temperature has not been achieved because the CaOx/O2 chemistry typically involves inert discharge products and few electrolytes can accommodate both a highly reductive Ca metal anode and O2. Here we report a Ca-O2 battery that is rechargeable for 700 cycles at room temperature. Our battery relies on a highly reversible two-electron redox to form chemically reactive calcium peroxide (CaO2) as the discharge product. Using a durable ionic liquid-based electrolyte, this two-electron reaction is enabled by the facilitated Ca plating-stripping in the Ca metal anode at room temperature and improved CaO2/O2 redox in the air cathode. We show the proposed Ca-O2 battery is stable in air and can be made into flexible fibres that are weaved into textile batteries for next-generation wearable systems.
RESUMEN
Serotonin (or 5-hydroxytryptamine, 5-HT) is an important neurotransmitter that activates 12 different G protein-coupled receptors (GPCRs) through selective coupling of Gs, Gi, or Gq proteins. The structural basis for G protein subtype selectivity by these GPCRs remains elusive. Here, we report the structures of the serotonin receptors 5-HT4, 5-HT6, and 5-HT7 with Gs, and 5-HT4 with Gi1. The structures reveal that transmembrane helices TM5 and TM6 alternate lengths as a macro-switch to determine receptor's selectivity for Gs and Gi, respectively. We find that the macro-switch by the TM5-TM6 length is shared by class A GPCR-G protein structures. Furthermore, we discover specific residues within TM5 and TM6 that function as micro-switches to form specific interactions with Gs or Gi. Together, these results present a common mechanism of Gs versus Gi protein coupling selectivity or promiscuity by class A GPCRs and extend the basis of ligand recognition at serotonin receptors.
Asunto(s)
Receptores Acoplados a Proteínas G , Serotonina , Proteínas de Unión al GTP/metabolismo , Ligandos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismoRESUMEN
Phosphorylation of G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) desensitizes G-protein signalling and promotes arrestin signalling, which is also modulated by biased ligands1-6. The molecular assembly of GRKs on GPCRs and the basis of GRK-mediated biased signalling remain largely unknown owing to the weak GPCR-GRK interactions. Here we report the complex structure of neurotensin receptor 1 (NTSR1) bound to GRK2, Gαq and the arrestin-biased ligand SBI-5537. The density map reveals the arrangement of the intact GRK2 with the receptor, with the N-terminal helix of GRK2 docking into the open cytoplasmic pocket formed by the outward movement of the receptor transmembrane helix 6, analogous to the binding of the G protein to the receptor. SBI-553 binds at the interface between GRK2 and NTSR1 to enhance GRK2 binding. The binding mode of SBI-553 is compatible with arrestin binding but clashes with the binding of Gαq protein, thus providing a mechanism for its arrestin-biased signalling capability. In sum, our structure provides a rational model for understanding the details of GPCR-GRK interactions and GRK2-mediated biased signalling.
Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G , Receptores Acoplados a Proteínas G , Transducción de Señal , Arrestinas/metabolismo , Fosforilación , Receptores Acoplados a Proteínas G/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/biosíntesis , Quinasa 2 del Receptor Acoplado a Proteína-G/química , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Ligandos , Unión Proteica , Receptores de Neurotensina/metabolismoRESUMEN
The dopamine system, including five dopamine receptors (D1R-D5R), plays essential roles in the central nervous system (CNS), and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders. Here, we report two cryo-EM structures of human D3R in complex with an inhibitory G protein and bound to the D3R-selective agonists PD128907 and pramipexole, the latter of which is used to treat patients with Parkinson's disease. The structures reveal agonist binding modes distinct from the antagonist-bound D3R structure and conformational signatures for ligand-induced receptor activation. Mutagenesis and homology modeling illuminate determinants of ligand specificity across dopamine receptors and the mechanisms for Gi protein coupling. Collectively our work reveals the basis of agonist binding and ligand-induced receptor activation and provides structural templates for designing specific ligands to treat CNS diseases targeting the dopaminergic system.
Asunto(s)
Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Modelos Moleculares , Complejos Multiproteicos/ultraestructura , Receptores de Dopamina D3/química , Benzopiranos/química , Células HEK293 , Humanos , Complejos Multiproteicos/química , Oxazinas/química , Pramipexol/química , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
Energy stress, characterized by the reduction of intracellular ATP, has been implicated in various diseases, including cancer. Here, we show that energy stress promotes the formation of P-bodies in a ubiquitin-dependent manner. Upon ATP depletion, the E3 ubiquitin ligase TRIM23 catalyzes lysine-63 (K63)-linked polyubiquitination of HCLS1-associated protein X-1 (HAX1). HAX1 ubiquitination triggers its liquidâliquid phase separation (LLPS) and contributes to P-bodies assembly induced by energy stress. Ubiquitinated HAX1 also interacts with the essential P-body proteins, DDX6 and LSM14A, promoting their condensation. Moreover, we find that this TRIM23/HAX1 pathway is critical for the inhibition of global protein synthesis under energy stress conditions. Furthermore, high HAX1 ubiquitination, and increased cytoplasmic localization of TRIM23 along with elevated HAX1 levels, promotes colorectal cancer (CRC)-cell proliferation and correlates with poor prognosis in CRC patients. Our data not only elucidate a ubiquitination-dependent LLPS mechanism in RNP granules induced by energy stress but also propose a promising target for CRC therapy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Lisina , Ubiquitinación , Humanos , Lisina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Estrés Fisiológico , Células HEK293 , Proliferación Celular , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión al GTPRESUMEN
Frontotemporal lobar degeneration (FTLD) is the third most common neurodegenerative condition after Alzheimer's and Parkinson's diseases1. FTLD typically presents in 45 to 64 year olds with behavioural changes or progressive decline of language skills2. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions with TAR DNA-binding protein (TDP-43) immunoreactivity3. Here we extracted amyloid fibrils from brains of four patients representing four of the five FTLD-TDP subclasses, and determined their structures by cryo-electron microscopy. Unexpectedly, all amyloid fibrils examined were composed of a 135-residue carboxy-terminal fragment of transmembrane protein 106B (TMEM106B), a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP4. In addition to TMEM106B fibrils, we detected abundant non-fibrillar aggregated TDP-43 by immunogold labelling. Our observations confirm that FTLD-TDP is associated with amyloid fibrils, and that the fibrils are formed by TMEM106B rather than TDP-43.
Asunto(s)
Amiloide , Proteínas de Unión al ADN , Degeneración Lobar Frontotemporal , Proteínas de la Membrana , Proteínas del Tejido Nervioso , Amiloide/ultraestructura , Microscopía por Crioelectrón , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Degeneración Lobar Frontotemporal/metabolismo , Degeneración Lobar Frontotemporal/patología , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/ultraestructuraRESUMEN
Thyroid-stimulating hormone (TSH), through activation of its G-protein-coupled thyrotropin receptor (TSHR), controls the synthesis of thyroid hormone-an essential metabolic hormone1-3. Aberrant signalling of TSHR by autoantibodies causes Graves' disease (hyperthyroidism) and hypothyroidism, both of which affect millions of patients worldwide4. Here we report the active structures of TSHR with TSH and the activating autoantibody M225, both bound to the allosteric agonist ML-1096, as well as an inactivated TSHR structure with the inhibitory antibody K1-707. Both TSH and M22 push the extracellular domain (ECD) of TSHR into an upright active conformation. By contrast, K1-70 blocks TSH binding and cannot push the ECD into the upright conformation. Comparisons of the active and inactivated structures of TSHR with those of the luteinizing hormone/choriogonadotropin receptor (LHCGR) reveal a universal activation mechanism of glycoprotein hormone receptors, in which a conserved ten-residue fragment (P10) from the hinge C-terminal loop mediates ECD interactions with the TSHR transmembrane domain8. One notable feature is that there are more than 15 cholesterols surrounding TSHR, supporting its preferential location in lipid rafts9. These structures also highlight a similar ECD-push mechanism for TSH and autoantibody M22 to activate TSHR, therefore providing the molecular basis for Graves' disease.
Asunto(s)
Inmunoglobulinas Estimulantes de la Tiroides , Receptores de Tirotropina , Tirotropina , Enfermedad de Graves/inmunología , Enfermedad de Graves/metabolismo , Humanos , Inmunoglobulinas Estimulantes de la Tiroides/inmunología , Microdominios de Membrana , Receptores de HL , Receptores de Tirotropina/agonistas , Receptores de Tirotropina/química , Receptores de Tirotropina/inmunología , Receptores de Tirotropina/metabolismo , Tirotropina/metabolismoRESUMEN
Adhesion G-protein-coupled receptors (aGPCRs) are important for organogenesis, neurodevelopment, reproduction and other processes1-6. Many aGPCRs are activated by a conserved internal (tethered) agonist sequence known as the Stachel sequence7-12. Here, we report the cryogenic electron microscopy (cryo-EM) structures of two aGPCRs in complex with Gs: GPR133 and GPR114. The structures indicate that the Stachel sequences of both receptors assume an α-helical-bulge-ß-sheet structure and insert into a binding site formed by the transmembrane domain (TMD). A hydrophobic interaction motif (HIM) within the Stachel sequence mediates most of the intramolecular interactions with the TMD. Combined with the cryo-EM structures, biochemical characterization of the HIM motif provides insight into the cross-reactivity and selectivity of the Stachel sequences. Two interconnected mechanisms, the sensing of Stachel sequences by the conserved 'toggle switch' W6.53 and the constitution of a hydrogen-bond network formed by Q7.49/Y7.49 and the P6.47/V6.47φφG6.50 motif (φ indicates a hydrophobic residue), are important in Stachel sequence-mediated receptor activation and Gs coupling. Notably, this network stabilizes kink formation in TM helices 6 and 7 (TM6 and TM7, respectively). A common Gs-binding interface is observed between the two aGPCRs, and GPR114 has an extended TM7 that forms unique interactions with Gs. Our structures reveal the detailed mechanisms of aGPCR activation by Stachel sequences and their Gs coupling.
Asunto(s)
Péptidos , Receptores Acoplados a Proteínas G , Sitios de Unión , Microscopía por Crioelectrón , Dominios Proteicos , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-ActividadRESUMEN
Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.
Asunto(s)
Receptores de Hormona Liberadora de Corticotropina/ultraestructura , Secuencia de Aminoácidos , Sitios de Unión , Hormona Liberadora de Corticotropina , Microscopía por Crioelectrón/métodos , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Péptidos/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Urocortinas/metabolismoRESUMEN
SNAI2/SLUG, a metastasis-promoting transcription factor, is a labile protein that is degraded through the ubiquitin proteasome degradation system. Here, we conducted comprehensive gain- and loss-of-function screens using a human DUB cDNA library of 65 genes and an siRNA library of 98 genes, and identified USP20 as a deubiquitinase (DUB) that regulates SNAI2 ubiquitination and stability. Further investigation of USP20 demonstrated its function in promoting migration, invasion, and metastasis of breast cancer. USP20 positively correlates with SNAI2 protein level in breast tumor samples, and higher USP20 expression is associated with poor prognosis in ER- breast cancer patients.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Metástasis de la Neoplasia/genética , Factores de Transcripción de la Familia Snail/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Neoplasias de la Mama/genética , Movimiento Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Biblioteca de Genes , Humanos , Invasividad Neoplásica/genética , Estabilidad Proteica , Proteolisis , ARN Interferente Pequeño/metabolismo , Ubiquitina Tiolesterasa/genética , UbiquitinaciónRESUMEN
Luteinizing hormone and chorionic gonadotropin are glycoprotein hormones that are related to follicle-stimulating hormone and thyroid-stimulating hormone1,2. Luteinizing hormone and chorionic gonadotropin are essential to human reproduction and are important therapeutic drugs3-6. They activate the same G-protein-coupled receptor, luteinizing hormone-choriogonadotropin receptor (LHCGR), by binding to the large extracellular domain3. Here we report four cryo-electron microscopy structures of LHCGR: two structures of the wild-type receptor in the inactive and active states; and two structures of the constitutively active mutated receptor. The active structures are bound to chorionic gonadotropin and the stimulatory G protein (Gs), and one of the structures also contains Org43553, an allosteric agonist7. The structures reveal a distinct 'push-and-pull' mechanism of receptor activation, in which the extracellular domain is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain. A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the interface between the extracellular domain and the transmembrane domain functions as a tethered agonist to induce conformational changes in the transmembrane domain and G-protein coupling. Org43553 binds to a pocket of the transmembrane domain and interacts directly with P10, which further stabilizes the active conformation. Together, these structures provide a common model for understanding the signalling of glycoprotein hormone receptors and a basis for drug discovery for endocrine diseases.
Asunto(s)
Receptores de HL/química , Gonadotropina Coriónica/química , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.
Asunto(s)
Microscopía por Crioelectrón , Ligandos , Lípidos , Receptores de Serotonina 5-HT1/metabolismo , Receptores de Serotonina 5-HT1/ultraestructura , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Aripiprazol/metabolismo , Aripiprazol/farmacología , Sitios de Unión , Colesterol/farmacología , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de Unión al GTP Heterotriméricas/ultraestructura , Humanos , Modelos Moleculares , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatos de Fosfatidilinositol/farmacología , Receptor de Serotonina 5-HT1A/química , Receptor de Serotonina 5-HT1A/metabolismo , Receptor de Serotonina 5-HT1A/ultraestructura , Receptores de Serotonina 5-HT1/química , Agonistas del Receptor de Serotonina 5-HT1/química , Agonistas del Receptor de Serotonina 5-HT1/metabolismo , Agonistas del Receptor de Serotonina 5-HT1/farmacología , Agua/químicaRESUMEN
Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available1-3. Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97)4-6, a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97-Go complexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the 'toggle switch' residue W6.53, which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the Go protein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the Go protein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling.