RESUMO
G-protein-coupled receptors (GPCRs) are a class of integral membrane proteins that detect environmental cues and trigger cellular responses. Deciphering the functional states of GPCRs induced by various ligands has been one of the primary goals in the field. Here we developed an effective universal method for GPCR cryo-electron microscopy structure determination without the need to prepare GPCR-signaling protein complexes. Using this method, we successfully solved the structures of the ß2-adrenergic receptor (ß2AR) bound to antagonistic and agonistic ligands and the adhesion GPCR ADGRL3 in the apo state. For ß2AR, an intermediate state stabilized by the partial agonist was captured. For ADGRL3, the structure revealed that inactive ADGRL3 adopts a compact fold and that large unusual conformational changes on both the extracellular and intracellular sides are required for activation of adhesion GPCRs. We anticipate that this method will open a new avenue for understanding GPCR structureâfunction relationships and drug development.
Assuntos
Receptores Adrenérgicos beta 2 , Receptores Acoplados a Proteínas G , Modelos Moleculares , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , LigantesRESUMO
Mas-related G-protein-coupled receptors X1-X4 (MRGPRX1-X4) are 4 primate-specific receptors that are recently reported to be responsible for many biological processes, including itch sensation, pain transmission, and inflammatory reactions. MRGPRX1 is the first identified human MRGPR, and its expression is restricted to primary sensory neurons. Due to its dual roles in itch and pain signaling pathways, MRGPRX1 has been regarded as a promising target for itch remission and pain inhibition. Here, we reported a cryo-electron microscopy (cryo-EM) structure of Gq-coupled MRGPRX1 in complex with a synthetic agonist compound 16 in an active conformation at an overall resolution of 3.0 Å via a NanoBiT tethering strategy. Compound 16 is a new pain-relieving compound with high potency and selectivity to MRGPRX1 over other MRGPRXs and opioid receptor. MRGPRX1 was revealed to share common structural features of the Gq-mediated receptor activation mechanism of MRGPRX family members, but the variable residues in orthosteric pocket of MRGPRX1 exhibit the unique agonist recognition pattern, potentially facilitating to design MRGPRX1-specific modulators. Together with receptor activation and itch behavior evaluation assays, our study provides a structural snapshot to modify therapeutic molecules for itch relieving and analgesia targeting MRGPRX1.
Assuntos
Prurido , Receptores Acoplados a Proteínas G , Animais , Humanos , Microscopia Crioeletrônica , Dor/metabolismo , Prurido/induzido quimicamente , Prurido/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de SinaisRESUMO
Yellow fever virus (YFV) infections can cause severe diseases in humans, resulting in mass casualties in Africa and the Americas each year. Secretory NS1 (sNS1) is thought to be used as a diagnostic marker of flavivirus infections, playing an essential role in the flavivirus life cycle, but little is known about the composition and structure of YFV sNS1. Here, we present that the recombinant YFV sNS1 exists in a heterogeneous mixture of oligomerizations, predominantly in the tetrameric form. The cryoEM structures show that the YFV tetramer of sNS1 is stacked by the hydrophobic interaction between ß-roll domains and greasy fingers. According to the 3D variability analysis, the tetramer is in a semi-stable state that may contain multiple conformations with dynamic changes. We believe that our study provides critical insights into the oligomerization of NS1 and will aid the development of NS1-based diagnoses and therapies.
Assuntos
Microscopia Crioeletrônica , Multimerização Proteica , Proteínas não Estruturais Virais , Vírus da Febre Amarela , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Vírus da Febre Amarela/química , Modelos Moleculares , Humanos , Conformação ProteicaRESUMO
Flaviviruses encode a conserved, membrane-associated nonstructural protein 1 (NS1) with replication and immune evasion functions. The current knowledge of secreted NS1 (sNS1) oligomers is based on several low-resolution structures, thus hindering the development of drugs and vaccines against flaviviruses. Here, we revealed that recombinant sNS1 from flaviviruses exists in a dynamic equilibrium of dimer-tetramer-hexamer states. Two DENV4 hexameric NS1 structures and several tetrameric NS1 structures from multiple flaviviruses were solved at atomic resolution by cryo-EM. The stacking of the tetrameric NS1 and hexameric NS1 is facilitated by the hydrophobic ß-roll and connector domains. Additionally, a triacylglycerol molecule located within the central cavity may play a role in stabilizing the hexamer. Based on differentiated interactions between the dimeric NS1, two distinct hexamer models (head-to-head and side-to-side hexamer) and the step-by-step assembly mechanisms of NS1 dimer into hexamer were proposed. We believe that our study sheds light on the understanding of the NS1 oligomerization and contributes to NS1-based therapies.
Assuntos
Microscopia Crioeletrônica , Flavivirus , Modelos Moleculares , Multimerização Proteica , Proteínas não Estruturais Virais , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Flavivirus/metabolismo , Flavivirus/química , Conformação ProteicaRESUMO
The chemotaxis of CD4+ type 1 helper cells and CD8+ cytotoxic lymphocytes, guided by interferon-inducible CXC chemokine 9-11 (CXCL9-11) and CXC chemokine receptor 3 (CXCR3), plays a critical role in type 1 immunity. Here we determined the structures of human CXCR3-DNGi complexes activated by chemokine CXCL11, peptidomimetic agonist PS372424 and biaryl-type agonist VUF11222, and the structure of inactive CXCR3 bound to noncompetitive antagonist SCH546738. Structural analysis revealed that PS372424 shares a similar orthosteric binding pocket to the N terminus of CXCL11, while VUF11222 buries deeper and activates the receptor in a distinct manner. We showed an allosteric binding site between TM5 and TM6, accommodating SCH546738 in the inactive CXCR3. SCH546738 may restrain the receptor at an inactive state by preventing the repacking of TM5 and TM6. By revealing the binding patterns and the pharmacological properties of the four modulators, we present the activation mechanisms of CXCR3 and provide insights for future drug development.
Assuntos
Quimiocinas CXC , Receptores CXCR3 , Humanos , Receptores CXCR3/metabolismo , Ligantes , Quimiocinas CXC/metabolismo , Sítios de Ligação , Ligação ProteicaRESUMO
The formyl peptide receptor 1 (FPR1) is primarily responsible for detection of short peptides bearing N-formylated methionine (fMet) that are characteristic of protein synthesis in bacteria and mitochondria. As a result, FPR1 is critical to phagocyte migration and activation in bacterial infection, tissue injury and inflammation. How FPR1 distinguishes between formyl peptides and non-formyl peptides remains elusive. Here we report cryo-EM structures of human FPR1-Gi protein complex bound to S. aureus-derived peptide fMet-Ile-Phe-Leu (fMIFL) and E. coli-derived peptide fMet-Leu-Phe (fMLF). Both structures of FPR1 adopt an active conformation and exhibit a binding pocket containing the R2015.38XXXR2055.42 (RGIIR) motif for formyl group interaction and receptor activation. This motif works together with D1063.33 for hydrogen bond formation with the N-formyl group and with fMet, a model supported by MD simulation and functional assays of mutant receptors with key residues for recognition substituted by alanine. The cryo-EM model of agonist-bound FPR1 provides a structural basis for recognition of bacteria-derived chemotactic peptides with potential applications in developing FPR1-targeting agents.
Assuntos
Moléculas com Motivos Associados a Patógenos , Staphylococcus aureus , Fatores Quimiotáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , N-Formilmetionina Leucil-Fenilalanina/química , Neutrófilos/metabolismo , Moléculas com Motivos Associados a Patógenos/metabolismo , Peptídeos/metabolismo , Staphylococcus aureus/metabolismoRESUMO
As a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to synthesize and translocate aminoacyl phospholipids to the outer leaflets of bacterial membranes. The function of MprFs enables Staphylococcus aureus and other pathogenic bacteria to acquire resistance to daptomycin and cationic antimicrobial peptides. Here we present cryo-electron microscopy structures of MprF homodimer from Rhizobium tropici (RtMprF) at two different states in complex with lysyl-phosphatidylglycerol (LysPG). RtMprF contains a membrane-embedded lipid-flippase domain with two deep cavities opening toward the inner and outer leaflets of the membrane respectively. Intriguingly, a hook-shaped LysPG molecule is trapped inside the inner cavity with its head group bent toward the outer cavity which hosts a second phospholipid-binding site. Moreover, RtMprF exhibits multiple conformational states with the synthase domain adopting distinct positions relative to the flippase domain. Our results provide a detailed framework for understanding the mechanisms of MprF-mediated modification and translocation of phospholipids.
Assuntos
Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilgliceróis/metabolismo , Fosfolipídeos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Transporte Biológico , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Lisina/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Fosfatidilgliceróis/química , Fosfolipídeos/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura , Rhizobium tropici/genética , Rhizobium tropici/metabolismoRESUMO
Overuse of ß2-adrenoceptor agonist bronchodilators evokes receptor desensitization, decreased efficacy, and an increased risk of death in asthma patients. Bronchodilators that do not target ß2-adrenoceptors represent a critical unmet need for asthma management. Here, we characterize the utility of osthole, a coumarin derived from a traditional Chinese medicine, in preclinical models of asthma. In mouse precision-cut lung slices, osthole relaxed preconstricted airways, irrespective of ß2-adrenoceptor desensitization. Osthole administered in murine asthma models attenuated airway hyperresponsiveness, a hallmark of asthma. Osthole inhibited phosphodiesterase 4D (PDE4D) activity to amplify autocrine prostaglandin E2 signaling in airway smooth muscle cells that eventually triggered cAMP/PKA-dependent relaxation of airways. The crystal structure of the PDE4D complexed with osthole revealed that osthole bound to the catalytic site to prevent cAMP binding and hydrolysis. Together, our studies elucidate a specific molecular target and mechanism by which osthole induces airway relaxation. Identification of osthole binding sites on PDE4D will guide further development of bronchodilators that are not subject to tachyphylaxis and would thus avoid ß2-adrenoceptor agonist resistance.
Assuntos
Asma , Cumarínicos , Animais , Asma/tratamento farmacológico , Cumarínicos/metabolismo , Cumarínicos/uso terapêutico , Medicamentos de Ervas Chinesas , Humanos , Pulmão/metabolismo , Camundongos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosforilação , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
In mitochondria, CDP-diacylglycerol (CDP-DAG) is a crucial precursor for cardiolipin biosynthesis. Mitochondrial CDP-DAG is synthesized by the translocator assembly and maintenance protein 41 (Tam41) through an elusive process. Here we show that Tam41 adopts sequential catalytic mechanism, and report crystal structures of the bulk N-terminal region of Tam41 from Schizosaccharomyces pombe in the apo and CTP-bound state. The structure reveals that Tam41 contains a nucleotidyltransferase (NTase) domain and a winged helix domain. CTP binds to an "L"-shaped pocket sandwiched between the two domains. Rearrangement of a loop region near the active site is essential for opening the CTP-binding pocket. Docking of phosphatidic acid/CDP-DAG in the structure suggests a lipid entry/exit pathway connected to the "L"-shaped pocket. The C-terminal region of SpTam41 contains a positively charged amphipathic helix crucial for membrane association and participates in binding phospholipids. These results provide detailed insights into the mechanism of CDP-DAG biosynthesis in mitochondria.