Asunto(s)
Escherichia coli/genética , Proteínas de Unión al GTP Heterotriméricas/genética , Clonación Molecular/métodos , Escherichia coli/crecimiento & desarrollo , Vectores Genéticos , Proteínas de Unión al GTP Heterotriméricas/aislamiento & purificación , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Isopropil Tiogalactósido/farmacología , Subunidades de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoAsunto(s)
Adenilil Ciclasas/aislamiento & purificación , Adenilil Ciclasas/química , Adenilil Ciclasas/genética , Animales , Perros , Escherichia coli/genética , Expresión Génica , Humanos , Membranas/enzimología , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , SolubilidadRESUMEN
Adenylyl cyclase (AC) is a prototypical cell-signaling molecule expressed in virtually all organisms from bacteria to man. While C1b, a poorly conserved region within mammalian AC, has been implicated in numerous isoform-specific regulatory properties, no one has purified the C1b region as a functional protein to homogeneity in order to study its role in enzyme function. We hypothesize that C1b is an internal regulatory subunit. To pursue this hypothesis, we constructed several soluble C1b proteins from type VII AC, arriving at one, 7C1b-S, which can be expressed and purified from Escherichia coli. 7C1b-S is relatively stable, as demonstrated by limited proteolytic analysis, circular dichroism, and UV Raman spectroscopy. Using size-exclusion chromatography and co-immunoprecipitation we demonstrate that 7C1b-S interacts with a cardinal activator of AC (Gsalpha) and with the conserved first catalytic domain (C1a) of type VII AC. We show that 7C1b-S inhibits Gsalpha-stimulated and Gsalpha-forskolin stimulated activity in our soluble ACVII model system. On the basis of these results, we suggest that 7C1b-S meets basic criteria to serve as a model protein for the C1b region and may be used as a prototype to develop other isoform C1b soluble model proteins to further investigate the role of this domain in isoform-specific regulation of adenylyl cyclase.
Asunto(s)
Adenilil Ciclasas/química , Adenilil Ciclasas/metabolismo , Citoplasma/enzimología , Fragmentos de Péptidos/química , Subunidades de Proteína/química , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/aislamiento & purificación , Secuencia de Aminoácidos , Dicroismo Circular , Secuencia Conservada , Citoplasma/metabolismo , Endopeptidasa K/metabolismo , Activadores de Enzimas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Hidrólisis , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , Solubilidad , Espectrometría RamanRESUMEN
Oedema factor, a calmodulin-activated adenylyl cyclase, is important in the pathogenesis of anthrax. Here we report the X-ray structures of oedema factor with and without bound calmodulin. Oedema factor shares no significant structural homology with mammalian adenylyl cyclases or other proteins. In the active site, 3'-deoxy-ATP and a single metal ion are well positioned for catalysis with histidine 351 as the catalytic base. This mechanism differs from the mechanism of two-metal-ion catalysis proposed for mammalian adenylyl cyclases. Four discrete regions of oedema factor form a surface that recognizes an extended conformation of calmodulin, which is very different from the collapsed conformation observed in other structures of calmodulin bound to effector peptides. On calmodulin binding, an oedema factor helical domain of relative molecular mass 15,000 undergoes a 15 A translation and a 30 degrees rotation away from the oedema factor catalytic core, which stabilizes a disordered loop and leads to enzyme activation. These allosteric changes provide the first molecular details of how calmodulin modulates one of its targets.