RESUMEN
Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.
Asunto(s)
Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Receptor del Glutamato Metabotropico 5/agonistas , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Agonistas de Aminoácidos Excitadores/metabolismo , Antagonistas de Aminoácidos Excitadores/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Subunidades de Proteína , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/ultraestructura , Relación Estructura-ActividadRESUMEN
The metabotropic glutamate (mGlu) receptors are class C G protein-coupled receptors (GPCRs) that modulate synaptic activity and plasticity throughout the mammalian brain. Signal transduction is initiated by glutamate binding to the venus flytrap domains (VFT), which initiates a conformational change that is transmitted to the conserved heptahelical domains (7TM) and results ultimately in the activation of intracellular G proteins. While both mGlu1 and mGlu5 activate Gαq G-proteins, they also increase intracellular cAMP concentration through an unknown mechanism. To study directly the G protein coupling properties of the human mGlu5 receptor homodimer, we purified the full-length receptor, which required careful optimisation of the expression, N-glycosylation and purification. We successfully purified functional mGlu5 that activated the heterotrimeric G protein Gq. The high-affinity agonist-PAM VU0424465 also activated the purified receptor in the absence of an orthosteric agonist. In addition, it was found that purified mGlu5 was capable of activating the G protein Gs either upon stimulation with VU0424465 or glutamate, although the later induced a much weaker response. Our findings provide important mechanistic insights into mGlu5 G protein-dependent activity and selectivity.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Animales , Línea Celular , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Glicosilación , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Receptor del Glutamato Metabotropico 5/química , Receptor del Glutamato Metabotropico 5/aislamiento & purificación , Solubilidad , Relación Estructura-ActividadRESUMEN
The need for early-on diagnostic tools to assess the folding and solubility of expressed protein constructs in vivo is of great interest when dealing with recalcitrant proteins. In this paper, we took advantage of the picomolar sensitivity of the bipartite GFP1-10/GFP11 system to investigate the solubility of the Mycobacterium tuberculosis 4'-phosphopantetheinyl transferase PptT, an enzyme essential for the viability of the tubercle bacillus. In vivo and in vitro complementation assays clearly showed the improved solubility of the full-length PptT compared to its N- and C-terminally truncated counterparts. However, initial attempts to purify the full-length enzyme overexpressed in Escherichia coli cells were hampered by aggregation issues overtime that caused the protein to precipitate within hours. The fact that the naturally occurring Coenzyme A and Mg(2+), essentials for PptT to carry out its function, could play a role in stabilizing the enzyme was confirmed using DSF experiments. In vitro activity assays were performed using the ACP substrate from the type I polyketide synthase PpsC from M. tuberculosis, a 2188 amino-acid enzyme that plays a major role in the virulence and pathogenicity of this microbial pathogen. We selected the most soluble and compact ACP fragment (2042-2188), identified by genetic selection of in-frame fragments from random library experiments, to monitor the transfer of the P-pant moiety from Coenzyme A onto a conserved serine residue of this ACP domain.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Mycobacterium tuberculosis/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/biosíntesis , Proteína Transportadora de Acilo/química , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Coenzima A/química , Estabilidad de Enzimas , Escherichia coli , Expresión Génica , Proteínas Fluorescentes Verdes/biosíntesis , Magnesio/química , Pliegue de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Solubilidad , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/aislamiento & purificaciónRESUMEN
In wheat, the high-molecular weight (HMW) glutenin subunits are known to contribute to gluten viscoelasticity, and show some similarities to elastomeric animal proteins as elastin. When combining the sequence of a glutenin with that of elastin is a way to create new chimeric functional proteins, which could be expressed in plants. The sequence of a glutenin subunit was modified by the insertion of several hydrophobic and elastic motifs derived from elastin (elastin-like peptide, ELP) into the hydrophilic repetitive domain of the glutenin subunit to create a triblock protein, the objective being to improve the mechanical (elastomeric) properties of this wheat storage protein. In this study, we investigated an expression model system to analyze the expression and trafficking of the wild-type HMW glutenin subunit (GS(W)) and an HMW glutenin subunit mutated by the insertion of elastin motifs (GS(M)-ELP). For this purpose, a series of constructs was made to express wild-type subunits and subunits mutated by insertion of elastin motifs in fusion with green fluorescent protein (GFP) in tobacco BY-2 cells. Our results showed for the first time the expression of HMW glutenin fused with GFP in tobacco protoplasts. We also expressed and localized the chimeric protein composed of plant glutenin and animal elastin-like peptides (ELP) in BY-2 protoplasts, and demonstrated its presence in protein body-like structures in the endoplasmic reticulum. This work, therefore, provides a basis for heterologous production of the glutenin-ELP triblock protein to characterize its mechanical properties.