RESUMEN
G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit1. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory Gs protein in complex with the ß2-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.
Asunto(s)
Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gs , Receptores Adrenérgicos beta 2 , Humanos , Sitios de Unión , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/ultraestructura , Factores de Tiempo , Activación Enzimática/efectos de los fármacos , Dominios Proteicos , Estructura Secundaria de Proteína , Transducción de Señal/efectos de los fármacosRESUMEN
The possible effects of mutations on stability and function of a protein can only be understood in the context of protein 3D structure. The MutationExplorer webserver maps sequence changes onto protein structures and allows users to study variation by inputting sequence changes. As the user enters variants, the 3D model evolves, and estimated changes in energy are highlighted. In addition to a basic per-residue input format, MutationExplorer can also upload an entire replacement sequence. Previously the purview of desktop applications, such an upload can back-mutate PDB structures to wildtype sequence in a single step. Another supported variation source is human single nucelotide polymorphisms (SNPs), genomic coordinates input in VCF format. Structures are flexibly colorable, not only by energetic differences, but also by hydrophobicity, sequence conservation, or other biochemical profiling. Coloring by interface score reveals mutation impacts on binding surfaces. MutationExplorer strives for efficiency in user experience. For example, we have prepared 45 000 PDB depositions for instant retrieval and initial display. All modeling steps are performed by Rosetta. Visualizations leverage MDsrv/Mol*. MutationExplorer is available at: http://proteinformatics.org/mutation_explorer/.
Asunto(s)
Internet , Modelos Moleculares , Mutación , Conformación Proteica , Proteínas , Programas Informáticos , Proteínas/genética , Proteínas/química , Humanos , Gráficos por Computador , TermodinámicaRESUMEN
The possible effects of mutations on stability and function of a protein can only be understood in the context of protein 3D structure. The MutationExplorer webserver maps sequence changes onto protein structures and allows users to study variation by inputting sequence changes. As the user enters variants, the 3D model evolves, and estimated changes in energy are highlighted. In addition to a basic per-residue input format, MutationExplorer can also upload an entire replacement sequence. Previously the purview of desktop applications, such an upload can back-mutate PDB structures to wildtype sequence in a single step. Another supported variation source is human single nucelotide polymorphisms (SNPs), genomic coordinates input in VCF format.
RESUMEN
Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. Here, we characterized the orphan aGPCR CG11318/mayo of Drosophila melanogaster and found it expressed in specific regions of the gastrointestinal canal and anal plates, epithelial specializations that control ion homeostasis. Genetic removal of mayo results in tachycardia, which is caused by hyperkalemia of the larval hemolymph. The hyperkalemic effect can be mimicked by a raise in ambient potassium concentration, while normal potassium levels in mayoKO mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development.
Asunto(s)
Drosophila , Hiperpotasemia , Animales , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Larva/metabolismo , Potasio/metabolismo , Taquicardia , Adhesión CelularRESUMEN
G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting guanine nucleotide exchange. Here, we investigate the coupling of G proteins with GPCRs and describe the events that ultimately lead to the ejection of GDP from its binding pocket in the Gα subunit, the rate-limiting step during G-protein activation. Using molecular dynamics simulations, we investigate the temporal progression of structural rearrangements of GDP-bound Gs protein (Gs·GDP; hereafter GsGDP) upon coupling to the ß2-adrenergic receptor (ß2AR) in atomic detail. The binding of GsGDP to the ß2AR is followed by long-range allosteric effects that significantly reduce the energy needed for GDP release: the opening of α1-αF helices, the displacement of the αG helix and the opening of the α-helical domain. Signal propagation to the Gs occurs through an extended receptor interface, including a lysine-rich motif at the intracellular end of a kinked transmembrane helix 6, which was confirmed by site-directed mutagenesis and functional assays. From this ß2AR-GsGDP intermediate, Gs undergoes an in-plane rotation along the receptor axis to approach the ß2AR-Gsempty state. The simulations shed light on how the structural elements at the receptor-G-protein interface may interact to transmit the signal over 30 Å to the nucleotide-binding site. Our analysis extends the current limited view of nucleotide-free snapshots to include additional states and structural features responsible for signaling and G-protein coupling specificity.