RESUMEN
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
In Arabidopsis thaliana, the acyl acid amido synthetase Gretchen Hagen 3.5 (AtGH3.5) conjugates both indole-3-acetic acid (IAA) and salicylic acid (SA) to modulate auxin and pathogen response pathways. To understand the molecular basis for the activity of AtGH3.5, we determined the X-ray crystal structure of the enzyme in complex with IAA and AMP. Biochemical analysis demonstrates that the substrate preference of AtGH3.5 is wider than originally described and includes the natural auxin phenylacetic acid (PAA) and the potential SA precursor benzoic acid (BA). Residues that determine IAA versus BA substrate preference were identified. The dual functionality of AtGH3.5 is unique to this enzyme although multiple IAA-conjugating GH3 proteins share nearly identical acyl acid binding sites. In planta analysis of IAA, PAA, SA, and BA and their respective aspartyl conjugates were determined in wild-type and overexpressing lines of A thaliana This study suggests that AtGH3.5 conjugates auxins (i.e., IAA and PAA) and benzoates (i.e., SA and BA) to mediate crosstalk between different metabolic pathways, broadening the potential roles for GH3 acyl acid amido synthetases in plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ácidos Indolacéticos/metabolismo , Ligasas/genética , Aminoácidos/química , Aminoácidos/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Cristalografía por Rayos X , Regulación de la Expresión Génica de las Plantas , Homeostasis , Ligasas/química , Ligasas/metabolismo , Ácido Salicílico/metabolismo , Especificidad por SustratoRESUMEN
In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 (SEP3), which is involved in many aspects of plant reproduction, including floral meristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening (I) and keratin-like (K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5-Å crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Proteínas de Dominio MADS/química , Proteínas de Dominio MADS/metabolismo , Multimerización de Proteína , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Cromatografía en Gel , Cristalografía por Rayos X , ADN de Plantas/metabolismo , Microscopía de Fuerza Atómica , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
The combination of protein crystallography and small-angle X-ray scattering (SAXS) provides a powerful method to investigate changes in protein conformation. These complementary structural techniques were used to probe the solution structure of the apo and the ligand-bound forms of the Arabidopsis thaliana acyl acid-amido synthetase GH3.12. This enzyme is part of the extensive GH3 family and plays a critical role in the regulation of plant hormones through the formation of amino-acid-conjugated hormone products via an ATP-dependent reaction mechanism. The enzyme adopts two distinct C-terminal domain orientations with `open' and `closed' active sites. Previous studies suggested that ATP only binds in the open orientation. Here, the X-ray crystal structure of GH3.12 is presented in the closed conformation in complex with the nonhydrolysable ATP analogue AMPCPP and the substrate salicylate. Using on-line HPLC purification combined with SAXS measurements, the most likely apo and ATP-bound protein conformations in solution were determined. These studies demonstrate that the C-terminal domain is flexible in the apo form and favours the closed conformation upon ATP binding. In addition, these data illustrate the efficacy of on-line HPLC purification integrated into the SAXS sample-handling environment to reliably monitor small changes in protein conformation through the collection of aggregate-free and highly redundant data.