RESUMEN
The bacterial enzyme, glycerol-3-phosphate cytidylyltransferase (GCT), is a model for mammalian cytidylyltransferases and is a member of a large superfamily of nucleotidyltransferases. Dimeric GCT from Bacillus subtilis displays unusual negative cooperativity in substrate binding and appears to form products only when both active sites are occupied by substrates. Here we describe a complex of GCT with the product, CDP-glycerol, in a crystal structure in which bound sulfate serves as a partial mimic of the second product, pyrophosphate. Binding of sulfate to form a pseudo-ternary complex is observed in three of the four chains constituting the asymmetric unit and is accompanied by a backbone rearrangement at Asp11 and ordering of the C-terminal helix. Comparison with the CTP complex of GCT, determined previously, reveals that in the product complex the active site closes around the glycerol phosphate moiety with a concerted motion of the segment 37-47 that includes helix B. This rearrangement allows lysines 44 and 46 to interact with the glycerol and cytosine phosphates of CDP-glycerol. Binding of CDP-glycerol also induces smaller movements of residues 92-100. Roles of lysines 44 and 46 in catalysis have been confirmed by mutagenesis of these residues to alanine, which decreases Vmax(app) and has profound effects on the Km(app) for glycerol-3-phosphate.
Asunto(s)
Proteínas Bacterianas/química , Nucleotidiltransferasas/química , Regulación Alostérica , Sustitución de Aminoácidos , Bacillus subtilis/enzimología , Catálisis , Cinética , Azúcares de Nucleósido Difosfato/química , Unión Proteica , Estructura Secundaria de Proteína , Sulfatos/químicaRESUMEN
The basic motif-leucine zipper (bZIP) transcription factor NRL controls the expression of rhodopsin and other phototransduction genes and is a key mediator of photoreceptor differentiation. To delineate the molecular mechanisms underlying transcriptional initiation of rod-specific genes, we characterized different regions of the NRL protein using yeast-based autoactivation assays. We identified 35 amino acid residues in the proline- and serine-rich N-terminal region (called minimal transactivation domain, MTD), which, when combined with LexA or Gal4 DNA binding domains, exhibited activation of target promoters. Because this domain is conserved in all proteins of the large Maf family, we hypothesized that NRL-MTD played an important role in assembling the transcription initiation complex. Our studies showed that the NRL protein, including the MTD, interacted with full-length or the C-terminal domain of TATA-binding protein (TBP) in vitro. NRL and TBP could be co-immunoprecipitated from bovine retinal nuclear extract. TBP was also part of c-Maf and MafA (two other large Maf proteins)-containing complex(es) in vivo. Our data suggest that the function of NRL-MTD is to activate transcription by recruiting or stabilizing TBP (and consequently other components of the general transcription complex) at the promoter of target genes, and a similar function may be attributed to other bZIP proteins of the large Maf family.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas del Ojo/química , Proteínas del Ojo/genética , Proteína de Unión a TATA-Box/química , Activación Transcripcional , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Bovinos , Núcleo Celular/metabolismo , Cristalografía por Rayos X , Cartilla de ADN/química , ADN Complementario/metabolismo , Activación Enzimática , Humanos , Inmunoprecipitación , Leucina Zippers , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Estructura Terciaria de Proteína , Retina/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Serina Endopeptidasas/química , Proteína de Unión a TATA-Box/metabolismo , Factores de Transcripción/química , Transcripción Genética , beta-Galactosidasa/metabolismoRESUMEN
Using peptides that represent linear regions of the powerful complement activation product, C5a, or loops that connect the four alpha helices of C5a, we have defined the ability of these peptides to reduce binding of (125)I-C5a to human neutrophils, inhibit chemotactic responses of neutrophils to C5a, and reduce H(2)O(2) production in neutrophils stimulated with PMA. The data have defined likely sites of interaction of C5a with C5aR. The peptides had no functional activity per se on neutrophils and did not interfere with neutrophil responses to the unrelated chemotactic peptide, N-formyl-Met-Leu-Phe. Although previous data have suggested that there are two separate sites on C5a reactive with C5aR, the current data suggest that C5a interacts with C5aR in a manner that engages three discontinuous regions of C5a.