RESUMEN
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
Bispecific antibodies (BsAbs) are undergoing continued development for applications in oncology and autoimmune diseases. While increasing activity by having more than one targeting arm, most BsAb engineering employs single Fc engagement as monoclonal antibodies. Here, we designed a novel immunoglobulin gamma-1 (IgG1)-derived dual-Fc BsAb containing two Fc regions and two distinct asymmetric antigen binding arms comprising a Fab arm and another VHH domain. In conjunction with the knob-into-hole technology, dual-Fc BsAbs could be produced with a high yield and good stability. We explore how Fc engineering effects on dual-Fc constructs could boost the desired therapeutic efficacy. This new format enabled simultaneous bispecific binding to corresponding antigens. Furthermore, compared to the one-Fc control molecules, dual-Fc BsAbs were shown to increase the avidity-based binding to FcγRs to result in higher ADCC and ADCP activities by potent avidity via binding to two antigens and Fc receptors. Overall, this novel BsAb format with enhanced effector functionalities provides a new option for antibody-based immunotherapy.
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Anticuerpos Biespecíficos , Anticuerpos Biespecíficos/química , Fragmentos Fc de Inmunoglobulinas/genética , Anticuerpos MonoclonalesRESUMEN
Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone-releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to ß-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus ß-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward ß-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.
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Empalme Alternativo/genética , Variación Genética/genética , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Células Cultivadas , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/genética , Células PC-3 , Células Sf9 , Transducción de Señal/genética , beta-Arrestinas/genéticaRESUMEN
Cholecystokinin A receptor (CCKAR) belongs to family A G-protein-coupled receptors and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with a sulfated ligand and to couple with divergent G-protein subtypes, including Gs, Gi and Gq. However, the basis for G-protein coupling promiscuity and ligand recognition by CCKAR remains unknown. Here, we present three cryo-electron microscopy structures of sulfated CCK-8-activated CCKAR in complex with Gs, Gi and Gq heterotrimers, respectively. CCKAR presents a similar conformation in the three structures, whereas conformational differences in the 'wavy hook' of the Gα subunits and ICL3 of the receptor serve as determinants in G-protein coupling selectivity. Our findings provide a framework for understanding G-protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8.
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Colecistoquinina/química , Receptor de Colecistoquinina A/química , Receptores Acoplados a Proteínas G/química , Sincalida/análogos & derivados , Secuencia de Aminoácidos , Benzodiazepinonas/química , Microscopía por Crioelectrón , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Sincalida/química , Triazoles/químicaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Chemical genomics has been applied extensively to evaluate small molecules that modulate biological processes in Saccharomyces cerevisiae. Here, we use yeast as a surrogate system for studying compounds that are active against metazoan targets. Large-scale chemical-genetic profiling of thousands of synthetic and natural compounds from the Chinese National Compound Library identified those with high-confidence bioprocess target predictions. To discover compounds that have the potential to function like therapeutic agents with known targets, we also analyzed a reference library of approved drugs. Previously uncharacterized compounds with chemical-genetic profiles resembling existing drugs that modulate autophagy and Wnt/ß-catenin signal transduction were further examined in mammalian cells, and new modulators with specific modes of action were validated. This analysis exploits yeast as a general platform for predicting compound bioactivity in mammalian cells.
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Autofagia/efectos de los fármacos , Descubrimiento de Drogas , Saccharomyces cerevisiae/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Correlación de Datos , Perfil Genético , Genómica/métodos , Células HEK293 , Células HeLa , Humanos , Prueba de Estudio Conceptual , beta Catenina/metabolismoRESUMEN
Gene-based molecular markers are increasingly used in crop breeding programs for marker-assisted selection. However, identification of genetic variants associated with important agronomic traits has remained a difficult task in soybean. RNA-Seq provides an efficient way, other than assessing global expression variations of coding genes, to discover gene-based SNPs at the whole genome level. In this study, RNA isolated from four soybean accessions each with three replications was subjected to high-throughput sequencing and a range of 44.2-65.9 million paired-end reads were generated for each library. A total of 75,209 SNPs were identified among different genotypes after combination of replications, 89.1% of which were located in expressed regions and 27.0% resulted in amino acid changes. GO enrichment analysis revealed that most significant enriched genes with nonsynonymous SNPs were involved in ribonucleotide binding or catalytic activity. Of 22 SNPs subjected to PCR amplification and Sanger sequencing, all of them were validated. To test the utility of identified SNPs, these validated SNPs were also assessed by genotyping a relative large population with 393 wild and cultivated soybean accessions. These SNPs identified by RNA-Seq provide a useful resource for genetic and genomic studies of soybean. Moreover, the collection of nonsynonymous SNPs annotated with their predicted functional effects also provides a valuable asset for further discovery of genes, identification of gene variants, and development of functional markers.
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Perfilación de la Expresión Génica/métodos , Glycine max/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Polimorfismo de Nucleótido Simple , Genotipo , Proteínas de Plantas/genética , Análisis de Secuencia de ARN/métodos , Glycine max/clasificaciónRESUMEN
Mutagenized populations have provided important materials for introducing variation and identifying gene function in plants. In this study, an ethyl methanesulfonate (EMS)-induced soybean (Glycine max) population, consisting of 21,600 independent M2 lines, was developed. Over 1,000 M4 (5) families, with diverse abnormal phenotypes for seed composition, seed shape, plant morphology and maturity that are stably expressed across different environments and generations were identified. Phenotypic analysis of the population led to the identification of a yellow pigmentation mutant, gyl, that displayed significantly decreased chlorophyll (Chl) content and abnormal chloroplast development. Sequence analysis showed that gyl is allelic to MinnGold, where a different single nucleotide polymorphism variation in the Mg-chelatase subunit gene (ChlI1a) results in golden yellow leaves. A cleaved amplified polymorphic sequence marker was developed and may be applied to marker-assisted selection for the golden yellow phenotype in soybean breeding. We show that the newly developed soybean EMS mutant population has potential for functional genomics research and genetic improvement in soybean.
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Biblioteca de Genes , Glycine max/genética , Mutación/genética , Clorofila/metabolismo , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Metanosulfonato de Etilo , Genoma de Planta , Fenotipo , Proteínas de Plantas/metabolismo , Polimorfismo Genético , Polimorfismo de Nucleótido Simple/genética , Carácter Cuantitativo Heredable , Semillas/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Leucine-rich repeat receptor-like kinases (LRR-RLKs) constitute the largest subfamily of receptor-like kinases in plant. A number of reports have demonstrated that plant LRR-RLKs play important roles in growth, development, differentiation, and stress responses. However, no comprehensive analysis of this gene family has been carried out in legume species. RESULTS: Based on the principles of sequence similarity and domain conservation, a total of 467 LRR-RLK genes were identified in soybean genome. The GmLRR-RLKs are non-randomly distributed across all 20 chromosomes of soybean and about 73.3 % of them are located in segmental duplicated regions. The analysis of synonymous substitutions for putative paralogous gene pairs indicated that most of these gene pairs resulted from segmental duplications in soybean genome. Furthermore, the exon/intron organization, motif composition and arrangements were considerably conserved among members of the same groups or subgroups in the constructed phylogenetic tree. The close phylogenetic relationship between soybean LRR-RLK genes with identified Arabidopsis genes in the same group also provided insight into their putative functions. Expression profiling analysis of GmLRR-RLKs suggested that they appeared to be differentially expressed among different tissues and some of duplicated genes exhibited divergent expression patterns. In addition, artificial selected GmLRR-RLKs were also identified by comparing the SNPs between wild and cultivated soybeans and 17 genes were detected in regions previously reported to contain domestication-related QTLs. CONCLUSIONS: Comprehensive and evolutionary analysis of soybean LRR-RLK gene family was performed at whole genome level. The data provides valuable tools in future efforts to identify functional divergence of this gene family and gene diversity among different genotypes in legume species.
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Evolución Molecular , Genoma de Planta , Glycine max/genética , Proteínas Quinasas/genética , Proteínas/genética , Arabidopsis/enzimología , Arabidopsis/genética , Secuencia Conservada , Duplicación de Gen , Proteínas Repetidas Ricas en Leucina , Familia de Multigenes , Motivos de Nucleótidos , Filogenia , Proteínas Quinasas/clasificación , Glycine max/enzimología , TranscriptomaRESUMEN
Metastasis of primary tumors remains a challenge for early diagnosis and prevention. The cellular properties and molecular drivers of metastatically competent clones within primary tumors remain unclear. Here, we generated 10-16 single cell-derived lines from each of three colorectal cancer (CRC) tumors to identify and characterize metastatic seeds. We found that intrinsic factors conferred clones with distinct metastatic potential and cellular communication capabilities, determining organ-specific metastasis. Poorly differentiated or highly metastatic clones, rather than drug-resistant clones, exhibited poor clinical prognostic impact. Personalized genetic alterations, instead of mutation burden, determined the occurrence of metastatic potential during clonal evolution. Additionally, we developed a gene signature for capturing metastatic potential of primary CRC tumors and demonstrated a strategy for identifying metastatic drivers using isogenic clones with distinct metastatic potential in primary tumors. This study provides insight into the origin and mechanisms of metastasis and will help develop potential anti-metastatic therapeutic targets for CRC patients.
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Carcinogénesis , Neoplasias Colorrectales , Humanos , Comunicación Celular , Línea Celular , Neoplasias Colorrectales/genética , SemillasRESUMEN
In recent years, the removal mechanism of ammonia nitrogen in black and odorous water (BOW), especially in the process of phytoremediation, has been a research "hotspot". Here, the migration process of ammonia nitrogen in macrophytes (Acorus calamus, Canna indica and Eichhornia crassipes) was detected by Fourier transform infrared (FT-IR) spectroscopy. Experiments revealed that the concentration of ammonia nitrogen (NH4 +-N) was reduced significantly. Maximum reduction in the NH4 +-N concentration was obtained in 75% BOW: the absorption of NH4 +-N was >90% in A. calamus and C. indica, and >80% in E. crassipes. After two 10 days cultivations, in the culture dishes of A. calamus and C. indica, absorption of NH4 +-N was >90% whereas, in the culture dishes of E. crassipes, absorption of NH4 +-N was â¼50% and â¼60%. FT-IR spectroscopy showed that NH4 +-N, NO2 --N and NO3 --N could be absorbed by the root and migrate to the stem and leaf of macrophytes. NH4 +-N and NO2 --N were transformed, and the direction was NH4 +-N â NO2 --N â NO3 --N. The migration rate of NH4 +-N in C. indica was faster because of its regular and smooth capillaries according to scanning electron microscopy. Our study on the removal and transformation mechanism of ammonia nitrogen in BOW could be an important reference for other bodies of water.
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5-hydroxytryptamine receptor 5A (5-HT5A) belongs to the 5-HT receptor family and signals through the Gi/o protein. It is involved in nervous system regulation and an attractive target for the treatment of psychosis, depression, schizophrenia, and neuropathic pain. 5-HT5A is the only Gi/o-coupled 5-HT receptor subtype lacking a high-resolution structure, which hampers the mechanistic understanding of ligand binding and Gi/o coupling for 5-HT5A. Here we report a cryo-electron microscopy structure of the 5-HT5A-Gi complex bound to 5-Carboxamidotryptamine (5-CT). Combined with functional analysis, this structure reveals the 5-CT recognition mechanism and identifies the receptor residue at 6.55 as a determinant of the 5-CT selectivity for Gi/o-coupled 5-HT receptors. In addition, 5-HT5A shows an overall conserved Gi protein coupling mode compared with other Gi/o-coupled 5-HT receptors. These findings provide comprehensive insights into the ligand binding and G protein coupling of Gi/o-coupled 5-HT receptors and offer a template for the design of 5-HT5A-selective drugs.
RESUMEN
Pre-existing drug resistance and tumorigenicity of cancer cells are highly correlated with therapeutic failure and tumor growth. However, current cancer models are limited in their application to the study of intratumor functional heterogeneity in personalized oncology. Here, an innovative two-dimensional (2D) and three-dimensional (3D) model for patient-derived cancer cells (PDCCs) and air-liquid interface (ALI) organotypic culture is established from colorectal cancer (CRC). The PDCCs recapitulate the genomic landscape of their parental tumors with high efficiency, high proliferation rate, and long-term stability, while corresponding ALI organotypic cultures retain histological architecture of their original tumors. Interestingly, both 2D and 3D models maintain the transcriptomic profile of the corresponding primary tumors and display the same trend in response to 5-Fluoruracil, regardless of their difference in gene expression profiles. Furthermore, single-cell-derived clones() are efficiently established and pre-existing drug-resistant clones and highly tumorigenic clones within individual CRC tumors are identified. It is found that tumorigenic cancer cells do not necessarily possess the stem cells characteristics in gene expression. This study provides valuable platform and resource for exploring the molecular mechanisms underlying the pre-existing drug resistance and tumorigenicity in cancer cells, as well as for developing therapeutic targets specifically for pre-existing drug-resistant or highly tumorigenic clones.
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Neoplasias Colorrectales , Organoides , Carcinogénesis , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Fluorouracilo/uso terapéutico , HumanosRESUMEN
Bradykinin and kallidin are endogenous kinin peptide hormones that belong to the kallikrein-kinin system and are essential to the regulation of blood pressure, inflammation, coagulation and pain control. Des-Arg10-kallidin, the carboxy-terminal des-Arg metabolite of kallidin, and bradykinin selectively activate two G protein-coupled receptors, type 1 and type 2 bradykinin receptors (B1R and B2R), respectively. The hyperactivation of bradykinin receptors, termed 'bradykinin storm', is associated with pulmonary edema in COVID-19 patients, suggesting that bradykinin receptors are important targets for COVID-19 intervention. Here we report two G protein-coupled complex structures of human B1R and B2R bound to des-Arg10-kallidin and bradykinin, respectively. Combined with functional analysis, our structures reveal the mechanism of ligand selectivity and specific activation of the bradykinin receptor. These findings also provide a framework for guiding drug design targeting bradykinin receptors for the treatment of inflammation, cardiovascular disorders and COVID-19.
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Bradiquinina/metabolismo , COVID-19/patología , Calidina/metabolismo , Receptores de Bradiquinina/metabolismo , Microscopía por Crioelectrón , Activación Enzimática/fisiología , Humanos , Estructura Terciaria de Proteína , Edema Pulmonar/patología , Edema Pulmonar/virología , SARS-CoV-2RESUMEN
Melanocortins are peptide hormones critical for the regulation of stress response, energy homeostasis, inflammation, and skin pigmentation. Their functions are mediated by five G protein-coupled receptors (MC1R-MC5R), predominately through the stimulatory G protein (Gs). MC1R, the founding member of melanocortin receptors, is mainly expressed in melanocytes and is involved in melanogenesis. Dysfunction of MC1R is associated with the development of melanoma and skin cancer. Here we present three cryo-electron microscopy structures of the MC1R-Gs complexes bound to endogenous hormone α-MSH, a marketed drug afamelanotide, and a synthetic agonist SHU9119. These structures reveal the orthosteric binding pocket for the conserved HFRW motif among melanocortins and the crucial role of calcium ion in ligand binding. They also demonstrate the basis of differential activities among different ligands. In addition, unexpected interactions between MC1R and the Gß subunit were discovered from these structures. Together, our results elucidate a conserved mechanism of calcium-mediated ligand recognition, a specific mode of G protein coupling, and a universal activation pathway of melanocortin receptors.
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Calcio , Proteínas de Unión al GTP/metabolismo , Receptor de Melanocortina Tipo 1 , Calcio/metabolismo , Microscopía por Crioelectrón , Humanos , Melanocortinas , Melanocitos , Receptor de Melanocortina Tipo 1/metabolismoRESUMEN
The COVID-19 pandemic caused by nonstop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report that suramin, a 100-year-old drug, is a potent inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and acts by blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for treatment of COVID-19. The 2.6 Å cryo-electron microscopy structure of the viral RdRp bound to suramin reveals two binding sites. One site directly blocks the binding of the RNA template strand and the other site clashes with the RNA primer strand near the RdRp catalytic site, thus inhibiting RdRp activity. Suramin blocks viral replication in Vero E6 cells, although the reasons underlying this effect are likely various. Our results provide a structural mechanism for a nonnucleotide inhibitor of the SARS-CoV-2 RdRp.
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Antivirales/farmacología , ARN Polimerasa Dependiente de ARN de Coronavirus/antagonistas & inhibidores , ARN Polimerasa Dependiente de ARN de Coronavirus/química , Inhibidores Enzimáticos/farmacología , Suramina/farmacología , Animales , Antivirales/química , Antivirales/metabolismo , Sitios de Unión , Dominio Catalítico , Chlorocebus aethiops , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Microscopía por Crioelectrón , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Conformación Proteica , ARN Viral/química , ARN Viral/metabolismo , SARS-CoV-2/efectos de los fármacos , Suramina/química , Suramina/metabolismo , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
Growth hormone-releasing hormone (GHRH) regulates the secretion of growth hormone that virtually controls metabolism and growth of every tissue through its binding to the cognate receptor (GHRHR). Malfunction in GHRHR signaling is associated with abnormal growth, making GHRHR an attractive therapeutic target against dwarfism (e.g., isolated growth hormone deficiency, IGHD), gigantism, lipodystrophy and certain cancers. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GHRHR bound to its endogenous ligand and the stimulatory G protein at 2.6 Å. This high-resolution structure reveals a characteristic hormone recognition pattern of GHRH by GHRHR, where the α-helical GHRH forms an extensive and continuous network of interactions involving all the extracellular loops (ECLs), all the transmembrane (TM) helices except TM4, and the extracellular domain (ECD) of GHRHR, especially the N-terminus of GHRH that engages a broad set of specific interactions with the receptor. Mutagenesis and molecular dynamics (MD) simulations uncover detailed mechanisms by which IGHD-causing mutations lead to the impairment of GHRHR function. Our findings provide insights into the molecular basis of peptide recognition and receptor activation, thereby facilitating the development of structure-based drug discovery and precision medicine.
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Hormona Liberadora de Hormona del Crecimiento/química , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Receptores de Neuropéptido/química , Receptores de Neuropéptido/metabolismo , Receptores de Hormona Reguladora de Hormona Hipofisaria/química , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Enanismo Hipofisario/genética , Proteínas de Unión al GTP , Hormona Liberadora de Hormona del Crecimiento/deficiencia , Humanos , Simulación de Dinámica Molecular , Mutagénesis , Mutación , Conformación Proteica , Conformación Proteica en Hélice alfa , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Transducción de SeñalRESUMEN
The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo-electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.
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Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/química , Betacoronavirus/enzimología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Alanina/química , Alanina/metabolismo , Alanina/farmacología , Antivirales/metabolismo , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Betacoronavirus/fisiología , Dominio Catalítico , ARN Polimerasa Dependiente de ARN de Coronavirus , Microscopía por Crioelectrón , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Complejos Multiproteicos/química , Conformación Proteica , ARN Viral/química , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2 , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Glucagon-like peptides (GLP-1 and GLP-2) are two proglucagon-derived intestinal hormones that mediate distinct physiological functions through two related receptors (GLP-1R and GLP-2R) which are important drug targets for metabolic disorders and Crohn's disease, respectively. Despite great progress in GLP-1R structure determination, our understanding on the differences of peptide binding and signal transduction between these two receptors remains elusive. Here we report the electron microscopy structure of the human GLP-2R in complex with GLP-2 and a Gs heterotrimer. To accommodate GLP-2 rather than GLP-1, GLP-2R fine-tunes the conformations of the extracellular parts of transmembrane helices (TMs) 1, 5, 7 and extracellular loop 1 (ECL1). In contrast to GLP-1, the N-terminal histidine of GLP-2 penetrates into the receptor core with a unique orientation. The middle region of GLP-2 engages with TM1 and TM7 more extensively than with ECL2, and the GLP-2 C-terminus closely attaches to ECL1, which is the most protruded among 9 class B G protein-coupled receptors (GPCRs). Functional studies revealed that the above three segments of GLP-2 are essential for GLP-2 recognition and receptor activation, especially the middle region. These results provide new insights into the molecular basis of ligand specificity in class B GPCRs and may facilitate the development of more specific therapeutics.