RESUMO
Cholesterol molecules specifically bind to the resting αßTCR to inhibit cytoplasmic CD3ζ ITAM phosphorylation through sequestering the TCR-CD3 complex in an inactive conformation. The mechanisms of cholesterol-mediated inhibition of TCR-CD3 and its activation remain unclear. Here, we present cryoelectron microscopy structures of cholesterol- and cholesterol sulfate (CS)-inhibited TCR-CD3 complexes and an auto-active TCR-CD3 variant. The structures reveal that cholesterol molecules act like a latch to lock CD3ζ into an inactive conformation in the membrane. Mutations impairing binding of cholesterol molecules to the tunnel result in the movement of the proximal C terminus of the CD3ζ transmembrane helix, thereby activating the TCR-CD3 complex in human cells. Together, our data reveal the structural basis of TCR inhibition by cholesterol, illustrate how the cholesterol-binding tunnel is allosterically coupled to TCR triggering, and lay a foundation for the development of immunotherapies through directly targeting the TCR-CD3 complex.
Assuntos
Complexo Receptor-CD3 de Antígeno de Linfócitos T , Linfócitos T , Complexo CD3/genética , Complexo CD3/metabolismo , Colesterol/metabolismo , Microscopia Crioeletrônica , Humanos , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
Adhesion G-protein-coupled receptors (aGPCRs) play key roles in a diversity of physiologies. A hallmark of aGPCR activation is the removal of the inhibitory GAIN domain and the dipping of the cleaved stalk peptide into the ligand-binding pocket of receptors; however, the detailed mechanism remains obscure. Here, we present cryoelectron microscopy (cryo-EM) structures of ADGRL3 in complex with Gq, Gs, Gi, and G12. The structures reveal unique ligand-engaging mode, distinctive activation conformation, and key mechanisms of aGPCR activation. The structures also reveal the uncharted structural information of GPCR/G12 coupling. A comparison of Gq, Gs, Gi, and G12 engagements with ADGRL3 reveals the key determinant of G-protein coupling on the far end of αH5 of Gα. A detailed analysis of the engagements allows us to design mutations that specifically enhance one pathway over others. Taken together, our study lays the groundwork for understanding aGPCR activation and G-protein-coupling selectivity.
Assuntos
Proteínas de Ligação ao GTP , Receptores Acoplados a Proteínas G , Ligantes , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Ligação ao GTP/metabolismoRESUMO
Plasmodium falciparum multidrug resistance protein 1 (PfMDR1), an adenosine triphosphate (ATP)-binding cassette (ABC) transporter on the digestive vacuole (DV) membrane of the parasite, is associated with the resistance to antimalarial drugs. To understand the mechanisms of PfMDR1, we determined the cryo-electron microscopy structures of this transporter in different states. The transporter in the apo state shows an inward-facing conformation with a large cavity opening to the cytoplasm. Upon ATP binding and dimerization of the nucleotide-binding domains (NBDs), PfMDR1 displays an outward-facing conformation with a cavity toward the DV lumen. Drug resistance-associated mutations were investigated in both structures for their effects, and Y184F was identified as an allosteric activity-enhancing mutation. The amphiphilic substrate-binding site of PfMDR1 was revealed by the complex structure with the antimalarial drug mefloquine and confirmed by mutagenesis studies. Remarkably, a helical structure was found to hinder NBD dimerization and inhibit PfMDR1 activity. The location of this regulatory domain in the N terminus is different from the well-studied R domain in the internal linker region of other ABC transporter family members. The lack of the phosphorylation site of this domain also suggests a different regulation mechanism.
Assuntos
Antimaláricos , Malária Falciparum , Humanos , Plasmodium falciparum , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Microscopia Crioeletrônica , Antimaláricos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Resistência a Medicamentos/genética , Malária Falciparum/parasitologiaRESUMO
Sphingosine-1-phosphate receptor 1 (S1PR1) is a master regulator of lymphocyte egress from the lymph node and an established drug target for multiple sclerosis (MS). Mechanistically, therapeutic S1PR1 modulators activate the receptor yet induce sustained internalization through a potent association with ß-arrestin. However, a structural basis of biased agonism remains elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of Gi-bound S1PR1 in complex with S1P, fingolimod-phosphate (FTY720-P) and siponimod (BAF312). In combination with functional assays and molecular dynamics (MD) studies, we reveal that the ß-arrestin-biased ligands direct a distinct activation path in S1PR1 through the extensive interplay between the PIF and the NPxxY motifs. Specifically, the intermediate flipping of W2696.48 and the retained interaction between F2656.44 and N3077.49 are the key features of the ß-arrestin bias. We further identify ligand-receptor interactions accounting for the S1PR subtype specificity of BAF312. These structural insights provide a rational basis for designing novel signaling-biased S1PR modulators.
Assuntos
Cloridrato de Fingolimode , Esclerose Múltipla , Microscopia Crioeletrônica , Cloridrato de Fingolimode/farmacologia , Cloridrato de Fingolimode/uso terapêutico , Humanos , Esclerose Múltipla/tratamento farmacológico , Receptores de Esfingosina-1-Fosfato , beta-ArrestinasRESUMO
A cyanomethylation/cyclization of aryl acetylenes/ethylenes with bromoacetonitrile was finished in a photopromoted condition, which offers an efficient and mild protocol for the preparation of cyanomethylated 7- or 5-membered N-heterocycles with good yields. Meanwhile, trichloroacetonitrile was also compatible with this radical pathway. In addition, a variety of single-crystal X-ray diffraction measurements, scaled-up operations to 1 mmol, functional group transformations of final products, light on/off experiments, and even radial inhibition studies were smoothly performed in this tandem system.
RESUMO
A metal/peroxide-free involved simple cascade 6-exo-trig spirocyclization of tert-butyl nitrite with biaryl ynones has been finished, which resulted in various NO2-modified spiro[5,5]trienones with good regioselectivity/yields. A variety of scaled-up experiments, reduction/epoxidation operations, and mechanistic studies were performed to verify the merits and spirocyclization process of this radical system. Finally, the structure of the spirocycles was confirmed by single-crystal X-ray diffraction.
RESUMO
Free radical three-component nitration/spirocyclization of unsaturated sulfonamides/amides with tert-butyl nitrite was developed for the construction of diverse NO2-revised 4-azaspiro[4.5]decanes. This tandem system featured metal-free participation, simple operation, good selectivity/yields, and a green/low-cost O source. Meanwhile, one nitro-containing complex molecule and a scaled-up operation were performed well to test the synthetic potential of the cascade reaction. Isotopic labeling, radical inhibition experiments, and DFT analysis were carried out to gain insight into the reaction process.
RESUMO
Although GPR3 plays pivotal roles in both the nervous system and metabolic processes, such as cold-induced thermogenesis, its endogenous ligand remains elusive. Here, by combining structural approach (including cryo-electron microscopy), mass spectrometry analysis, and functional studies, we identify oleic acid (OA) as an endogenous ligand of GPR3. Our study reveals a hydrophobic tunnel within GPR3 that connects the extracellular side of the receptor to the middle of plasma membrane, enabling fatty acids to readily engage the receptor. Functional studies demonstrate that OA triggers downstream Gs signaling, whereas lysophospholipids fail to activate the receptor. Moreover, our research reveals that cold stimulation induces the secretion of OA in mice, subsequently activating Gs/cAMP/PKA signaling in brown adipose tissue. Notably, brown adipose tissues from Gpr3 knockout mice do not respond to OA during cold stimulation, reinforcing the significance of GPR3 in this process. Finally, we propose a "born to be activated and cold to enhance" model for GPR3 activation. Our study provides a starting framework for the understanding of GPR3 signaling in cold-stimulated thermogenesis.
Assuntos
Tecido Adiposo Marrom , Ácido Oleico , Receptores Acoplados a Proteínas G , Animais , Camundongos , Membrana Celular , Microscopia Crioeletrônica , Ligantes , Camundongos Knockout , Ácido Oleico/metabolismo , Ácido Oleico/farmacologia , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The histamine H4 receptor (H4R) plays key role in immune cell function and is a highly valued target for treating allergic and inflammatory diseases. However, structural information of H4R remains elusive. Here, we report four cryo-EM structures of H4R/Gi complexes, with either histamine or synthetic agonists clobenpropit, VUF6884 and clozapine bound. Combined with mutagenesis, ligand binding and functional assays, the structural data reveal a distinct ligand binding mode where D943.32 and a π-π network determine the orientation of the positively charged group of ligands, while E1825.46, located at the opposite end of the ligand binding pocket, plays a key role in regulating receptor activity. The structural insight into H4R ligand binding allows us to identify mutants at E1825.46 for which the agonist clobenpropit acts as an inverse agonist and to correctly predict inverse agonism of a closely related analog with nanomolar potency. Together with the findings regarding receptor activation and Gi engagement, we establish a framework for understanding H4R signaling and provide a rational basis for designing novel antihistamines targeting H4R.
Assuntos
Agonismo Inverso de Drogas , Histamina , Imidazóis , Tioureia/análogos & derivados , Histamina/metabolismo , Receptores Histamínicos H4 , Receptores Acoplados a Proteínas G/metabolismo , Ligantes , Receptores Histamínicos/metabolismo , Antagonistas dos Receptores Histamínicos/farmacologiaRESUMO
CRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here we present a new family of RNA-guided DNA endonucleases. Our bioinformatics analysis initially identifies the stable co-occurrence of conserved RAGATH-18-derived RNAs (reRNAs) and their upstream IS607 TnpBs with an average length of 390 amino acids. IS607 TnpBs form programmable DNases through interaction with reRNAs. We discover the robust dsDNA interference activity of IS607 TnpB systems in bacteria and human cells. Further characterization of the Firmicutes bacteria IS607 TnpB system (ISFba1 TnpB) reveals that its dsDNA cleavage activity is remarkably sensitive to single mismatches between the guide and target sequences in human cells. Our findings demonstrate that a length of 20 nt in the guide sequence of reRNA achieves the highest DNA cleavage activity for ISFba1 TnpB. A cryo-EM structure of the ISFba1 TnpB effector protein bound by its cognate RAGATH-18 motif-containing reRNA and a dsDNA target reveals the mechanisms underlying reRNA recognition by ISFba1 TnpB, reRNA-guided dsDNA targeting, and the sensitivity of the ISFba1 TnpB system to base mismatches between the guide and target DNA. Collectively, this study identifies the IS607 TnpB family of compact and specific RNA-guided DNases with great potential for application in gene editing.
Assuntos
Sistemas CRISPR-Cas , Humanos , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , DNA/metabolismo , Edição de Genes , Endonucleases/metabolismo , Células HEK293 , Clivagem do DNARESUMO
CD97 (ADGRE5) is an adhesion G protein-coupled receptor (aGPCR) which plays crucial roles in immune system and cancer. However, the mechanism of CD97 activation and the determinant of G13 coupling selectivity remain unknown. Here, we present the cryo-electron microscopy structures of human CD97 in complex with G13, Gq, and Gs. Our structures reveal the stalk peptide recognition mode of CD97, adding missing information of the current tethered-peptide activation model of aGPCRs. For instance, a revised "FXφφφ" motif and a framework of conserved aromatic residues in the ligand-binding pocket. Importantly, structural comparisons of G13, Gq, and Gs engagements of CD97 reveal key determinants of G13 coupling selectivity, where a deep insertion of the α helix 5 and a closer contact with the transmembrane helix 6, 5, and 3 dictate coupling preferences. Taken together, our structural study of CD97 provides a framework for understanding CD97 signaling and the G13 coupling selectivity.
Assuntos
Proteínas de Ligação ao GTP , Receptores Acoplados a Proteínas G , Humanos , Microscopia Crioeletrônica , Peptídeos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
Lysophosphatidylserine (LysoPS) is a lipid mediator that induces multiple cellular responses through binding to GPR174. Here, we present the cryo-electron microscopy (cryo-EM) structure of LysoPS-bound human GPR174 in complex with Gs protein. The structure reveals a ligand recognition mode, including the negatively charged head group of LysoPS forms extensive polar interactions with surrounding key residues of the ligand binding pocket, and the L-serine moiety buries deeply into a positive charged cavity in the pocket. In addition, the structure unveils a partially open pocket on transmembrane domain helix (TM) 4 and 5 for a lateral entry of ligand. Finally, the structure reveals a Gs engaging mode featured by a deep insertion of a helix 5 (αH5) and extensive polar interactions between receptor and αH5. Taken together, the information revealed by our structural study provides a framework for understanding LysoPS signaling and a rational basis for designing LysoPS receptor-targeting drugs.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Ligantes , Microscopia CrioeletrônicaRESUMO
In the application of classical graph theory, there always are various indeterministic factors. This study studies the indeterministic factors in the connected graph by employing the uncertainty theory. First, this study puts forward two concepts: generalized uncertain graph and its connectivity index. Second, it presents a new algorithm to compute the connectivity index of an uncertain graph and generalized uncertain graph and verify this algorithm with typical examples. In addition, it proposes the definition and algorithm of α-connectivity index of generalized uncertain graph and verifies the stability and efficiency of this new algorithm by employing numerical experiments.
RESUMO
The B cell receptor (BCR) complex plays a critical role in B cell development and immune responses. The assembly mechanisms underlying the BCR complex remain unknown. We determined the cryo-electron microscopy (cryo-EM) structures of human IgG-BCR and IgM-BCR, which consist of membrane-bound immunoglobulin molecules (mIg) and Igα/ß subunits at a 1:1 stoichiometry. Assembly of both BCR complexes involves their extracellular domains, membrane-proximal connection peptides, and transmembrane (TM) helices. The TM helices of mIgG and mIgM share a conserved set of hydrophobic and polar interactions with Igα/ß TM helices. By contrast, the IgG-Cγ3 and IgM-Cµ4 domains interact with extracellular Ig-like domains of Igα/ß through head-to-tail and side-by-side modes, respectively. This work reveals the structural basis for BCR assembly and provides insights into BCR triggering.
Assuntos
Antígenos CD79 , Imunoglobulina G , Imunoglobulina M , Antígenos CD79/química , Microscopia Crioeletrônica , Humanos , Imunoglobulina G/química , Imunoglobulina M/química , Conformação Proteica em alfa-HéliceRESUMO
Adhesion G protein-coupled receptors (aGPCRs) are keys of many physiological events and attractive targets for various diseases. aGPCRs are also known to be capable of self-activation via an autoproteolysis process that removes the inhibitory GAIN domain on the extracellular side of receptor and releases a stalk peptide to bind and activate the transmembrane side of receptor. However, the detailed mechanism of aGPCR activation remains elusive. Here, we report the cryo-electron microscopy structures of GPR110 (ADGRF1), a member of aGPCR, in complex with Gq, Gs, Gi, G12 and G13. The structures reveal distinctive ligand engaging model and activation conformations of GPR110. The structures also unveil the rarely explored GPCR/G12 and GPCR/G13 engagements. A comparison of Gq, Gs, Gi, G12 and G13 engagements with GPR110 reveals details of G-protein engagement, including a dividing point at the far end of the alpha helix 5 (αH5) of Gα subunit that separates Gq/Gs engagements from Gi/G12/G13 engagements. This is also where Gq/Gs bind the receptor through both hydrophobic and polar interaction, while Gi/G12/G13 engage receptor mainly through hydrophobic interaction. We further provide physiological evidence of GPR110 activation via stalk peptide. Taken together, our study fills the missing information of GPCR/G-protein engagement and provides a framework for understanding aGPCR activation and GPR110 signaling.
Assuntos
Proteínas de Ligação ao GTP , Receptores Acoplados a Proteínas G , Adesão Celular , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/metabolismo , Ligantes , Peptídeos/química , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Histamine receptors play important roles in various pathophysiological conditions and are effective targets for anti-allergy treatment, however the mechanism of receptor activation remain elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human H1R in complex with a Gq protein in an active conformation via a NanoBiT tethering strategy. The structure reveals that histamine activates receptor via interacting with the key residues of both transmembrane domain 3 (TM3) and TM6 to squash the binding pocket on the extracellular side and to open the cavity on the intracellular side for Gq engagement in a model of "squash to activate and expand to deactivate". The structure also reveals features for Gq coupling, including the interaction between intracellular loop 2 (ICL2) and the αN-ß junction of Gq/11 protein. The detailed analysis of our structure will provide a framework for understanding G-protein coupling selectivity and clues for designing novel antihistamines.
Assuntos
Microscopia Crioeletrônica/métodos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Histamina/química , Receptores Histamínicos/química , Sítios de Ligação , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP , Histamina/metabolismo , Humanos , Ligantes , Ligação Proteica , Domínios Proteicos , Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/metabolismoRESUMO
Efficiency simplified: A synthetic strategy has been developed to prepare single-crystalline hollow Pd/Pt nanocubes (right, see picture; left: nanocubes). Compared to the solid Pd/Pt nanocubes of similar sizes, the hollow Pd/Pt nanocubes increase accessible surface area and therefore improve electrocatalytic activity in formic acid oxidation.