RESUMEN
Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants, fungi and bacteria. They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states, thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses. Several structures of microbial phytochromes have been determined in their dark-adapted Pr or Pfr states. However, the structural nature of initial photochemical events has not been characterized by crystallography. Here we report the crystal structures of three intermediates in the photoreaction of Pseudomonas aeruginosa bacteriophytochrome (PaBphP). We used cryotrapping crystallography to capture intermediates, and followed structural changes by scanning the temperature at which the photoreaction proceeded. Light-induced conformational changes in PaBphP originate in ring D of the biliverdin (BV) chromophore, and E-to-Z isomerization about the C(15) = C(16) double bond between rings C and D is the initial photochemical event. As the chromophore relaxes, the twist of the C(15) methine bridge about its two dihedral angles is reversed. Structural changes extend further to rings B and A, and to the surrounding protein regions. These data indicate that absorption of a photon by the Pfr state of PaBphP converts a light signal into a structural signal via twisting and untwisting of the methine bridges in the linear tetrapyrrole within the confined protein cavity.
Asunto(s)
Fitocromo/química , Fitocromo/metabolismo , Pseudomonas aeruginosa/química , Temperatura , Absorción , Biliverdina/química , Biliverdina/efectos de la radiación , Cristalografía , Isomerismo , Luz , Modelos Moleculares , Fosforilación , Procesos Fotoquímicos/efectos de la radiación , Fotones , Fitocromo/efectos de la radiación , Conformación Proteica/efectos de la radiación , TetrapirrolesRESUMEN
Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria by means of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here, we report the crystal structure of the Q188L mutant of Pseudomonas aeruginosa bacteriophytochrome (PaBphP) photosensory core module, which exhibits altered photoconversion behavior and different crystal packing from wild type. We observe two distinct chromophore conformations in the Q188L crystal structure that we identify with the Pfr and Pr states. The Pr/Pfr compositions, varying from crystal to crystal, seem to correlate with light conditions under which the Q188L crystals are cryoprotected. We also compare all known Pr and Pfr structures. Using site-directed mutagenesis, we identify residues that are involved in stabilizing the 15Ea (Pfr) and 15Za (Pr) configurations of the biliverdin chromophore. Specifically, Ser-261 appears to be essential to form a stable Pr state in PaBphP, possibly by means of its interaction with the propionate group of ring C. We propose a "flip-and-rotate" model that summarizes the major conformational differences between the Pr and Pfr states of the chromophore and its binding pocket.
Asunto(s)
Proteínas Bacterianas/química , Fotorreceptores Microbianos/química , Fitocromo/química , Pseudomonas aeruginosa/química , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Procesos Fotoquímicos , Fotorreceptores Microbianos/genética , Fitocromo/genética , Conformación Proteica , Pseudomonas aeruginosa/genética , Espectrofotometría , Electricidad EstáticaRESUMEN
Bacteriophytochromes (BphPs) are photoreceptors that regulate a wide range of biological mechanisms via red light-absorbing (Pr)-to-far-red light-absorbing (Pfr) reversible photoconversion. The structural dynamics underlying Pfr-to-Pr photoconversion in a liquid solution phase are not well understood. We used time-resolved x-ray solution scattering (TRXSS) to capture light-induced structural transitions in the bathy BphP photosensory module of Pseudomonas aeruginosa. Kinetic analysis of the TRXSS data identifies three distinct structural species, which are attributed to lumi-F, meta-F, and Pr, connected by time constants of 95 µs and 21 ms. Structural analysis based on molecular dynamics simulations shows that the light activation of PaBphP accompanies quaternary structural rearrangements from an "II"-framed close form of the Pfr state to an "O"-framed open form of the Pr state in terms of the helical backbones. This study provides mechanistic insights into how modular signaling proteins such as BphPs transmit structural signals over long distances and regulate their downstream biological responses.
RESUMEN
Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria via reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here we report the crystal structure at 2.9 A resolution of a bacteriophytochrome from Pseudomonas aeruginosa with an intact, fully photoactive photosensory core domain in its dark-adapted Pfr state. This structure reveals how unusual interdomain interactions, including a knot and an "arm" structure near the chromophore site, bring together the PAS (Per-ARNT-Sim), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA), and PHY (phytochrome) domains to achieve Pr/Pfr photoconversion. The PAS, GAF, and PHY domains have topologic elements in common and may have a single evolutionary origin. We identify key interactions that stabilize the chromophore in the Pfr state and provide structural and mutational evidence to support the essential role of the PHY domain in efficient Pr/Pfr photoconversion. We also identify a pair of conserved residues that may undergo concerted conformational changes during photoconversion. Modeling of the full-length bacteriophytochrome structure, including its output histidine kinase domain, suggests how local structural changes originating in the photosensory domain modulate interactions between long, cross-domain signaling helices at the dimer interface and are transmitted to the spatially distant effector domain, thereby regulating its histidine kinase activity.
Asunto(s)
Modelos Moleculares , Fitocromo/química , Fitocromo/metabolismo , Pseudomonas aeruginosa/química , Transducción de Señal , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Fotoquímica , Estructura Terciaria de ProteínaRESUMEN
RpBphP2 and RpBphP3, two tandem bacteriophytochromes from the photosynthetic bacterium Rhodopseudomonas palustris, share high sequence identity but exhibit distinct photoconversion behavior. Unlike the canonical RpBphP2, RpBphP3 photoconverts to an unusual near-red-absorbing (Pnr) state; both are required for synthesis of light-harvesting complexes under low-light conditions. Here we report the crystal structures of the photosensory core modules of RpBphP2 and RpBphP3. Despite different quaternary structures, RpBphP2 and RpBphP3 adopt nearly identical tertiary structures. The RpBphP3 structure reveals tongue-and-groove interactions at the interface between the GAF and PHY domains. A single mutation in the PRxSF motif at the GAF-PHY interface abolishes light-induced formation of the Pnr state in RpBphP3, possibly due to altered structural rigidity of the chromophore-binding pocket. Structural comparisons suggest that long-range signaling involves structural rearrangement of the helical spine at the dimer interface. These structures, together with mutational studies, provide insights into photoconversion and the long-range signaling mechanism in phytochromes.
Asunto(s)
Proteínas Bacterianas/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Fototransducción , Modelos Moleculares , Datos de Secuencia Molecular , Fitocromo/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Rhodopseudomonas/químicaRESUMEN
Structure-activity studies were performed on the alpha(1A)-adrenoceptor (AR) selective agonist N-[5-(1H-imidazol-4-yl)-5,6,7,8-tetrahydro-1-naphthalenyl]methanesulfonamide (4). Compounds were evaluated for binding activity at the alpha(1A), alpha(1b), alpha(1d), alpha(2a), and alpha(2B) subtypes. Functional activity in tissues containing the alpha(1A) (rabbit urethra), alpha(1B) (rat spleen), alpha(1D) (rat aorta), and alpha(2A) (rat prostatic vas deferens) was also evaluated. A dog in vivo model simultaneously measuring intraurethral pressure (IUP) and mean arterial pressure (MAP) was used to assess the uroselectivity of the compounds. Many of the compounds that were highly selective in vitro for the alpha(1A)-AR subtype were also more uroselective in vivo for increasing IUP over MAP than the nonselective alpha(1)-agonists phenylpropanolamine (PPA) (1) and ST-1059 (2, the active metabolite of midodrine), supporting the hypothesis that greater alpha(1A) selectivity would reduce cardiovascular side effects. However, the data also support a prominent role of the alpha(1A)-AR subtype in the control of MAP.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 1 , Imidazoles/síntesis química , Naftalenos/síntesis química , Sulfonamidas/síntesis química , Tetrahidronaftalenos/síntesis química , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Presión Sanguínea/efectos de los fármacos , Perros , Femenino , Imidazoles/química , Imidazoles/farmacología , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Naftalenos/química , Naftalenos/farmacología , Conejos , Ensayo de Unión Radioligante , Ratas , Receptores Adrenérgicos alfa 1 , Bazo/efectos de los fármacos , Bazo/fisiología , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , Tetrahidronaftalenos/química , Tetrahidronaftalenos/farmacología , Uretra/efectos de los fármacos , Uretra/fisiología , Conducto Deferente/efectos de los fármacos , Conducto Deferente/fisiologíaRESUMEN
Bacteriophytochromes RpBphP2 and RpBphP3 from the photosynthetic bacterium Rhodopseudomonas palustris work in tandem to modulate synthesis of the light-harvesting complex LH4 in response to light. Although RpBphP2 and RpBphP3 share the same domain structure with 52% sequence identity, they demonstrate distinct photoconversion behaviors. RpBphP2 exhibits the "classical" phytochrome behavior of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states, whereas RpBphP3 exhibits novel photoconversion between Pr and a near-red (Pnr) light-absorbing states. We have determined the crystal structure at 2.2-A resolution of the chromophore binding domains of RpBphP3, covalently bound with chromophore biliverdin IXalpha. By combining structural and sequence analyses with site-directed mutagenesis, we identify key residues that directly modulate the photochemical properties of RpBphP3 and RpBphP2. Remarkably, we identify a region spanning residues 207-212 in RpBphP3, in which a single mutation, L207Y, causes this unusual bacteriophytochrome to revert to the classical phenotype that undergoes reversible photoconversion between the Pr and Pfr states. The reverse mutation, Y193L, in the corresponding region in RpBphP2 significantly diminishes the formation of the Pfr state. We propose that residues 207-212 and the spatially adjacent conserved residues, Asp-216 and Tyr-272, interact with the chromophore and form part of the interface between the chromophore binding domains and the PHY domain that modulates photoconversion.
Asunto(s)
Biliverdina/química , Biliverdina/metabolismo , Fitocromo/química , Fitocromo/metabolismo , Rhodobacter/química , Rhodobacter/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Fotoquímica , Fitocromo/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Rhodobacter/genética , Alineación de SecuenciaRESUMEN
Glucocorticoids are potent anti-inflammatory and immunosuppressant agents. However, they also produce serious side effects that limit their usage. It has been proposed that anti-inflammatory properties of glucocorticoids are caused mostly by repression of activator protein 1- and nuclear factor kappabeta-stimulated synthesis of inflammatory mediators, whereas most of their adverse effects are associated with trans-activation of genes involved with metabolic processes. Our laboratories have sought to discover novel glucocorticoid receptor (GR) ligands that have high repression but low trans-activation activities. We describe here cellular properties of 2,5-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5-(1-methylcyclohexen-3-y1)-1H-[1]benzopyrano[3,4-f]quinoline (A276575) and its four enantiomers. Similar to dexamethasone, A276575 exhibited high affinity for GR and potently repressed interleukin (IL) 1beta-stimulated IL-6 production in human skin fibroblasts, prostaglandin (PG) E(2) production in A549 human lung epithelial cells, and concanavalin A-induced monocyte proliferation. In contrast to dexamethasone, A276575 caused smaller induction of aromatase activity in human skin fibroblasts and antagonized dexamethasone-induced activation of an mouse mammary tumor virus-glucocorticoid-response element (GRE) reporter gene construct. Among the four enantiomers of A276575, the two (-)-enantiomers showed 10- to 30-fold higher affinities for GR than their respective (+)-enantiomers. Both (-)-Syn and (-)-Anti enantiomers of A276575 were potent inhibitors of IL-1beta-stimulated PGE2 production in A549 lung epithelial cells; unexpectedly, however, only the (-)-Anti enantiomer inhibited regulated on T-cell activation, normal T-cell expressed and secreted (RANTES) production in A549 cells. In summary, A276575 is a novel, nonsteroidal GR ligand that possesses high repression activities against inflammatory mediator production but has lower GRE trans-activation activities than traditional steroids. Differential repression of RANTES and PGE2 production in a cell by the two (-)-enantiomers of A276575 illustrates the complexity of repression by GR.