RESUMEN
The anthracis repressor (AntR) is a manganese-activated transcriptional regulator from Bacillus anthracis and is a member of the diphtheria toxin repressor (DtxR) family of proteins. In this paper, we characterize the Mn(II) binding and protein dimerization state using a combination of continuous wave (cw) and pulsed EPR methods. Equilibrium metal binding experiments showed that AntR binds 2 equivalents of Mn(II) with positive cooperativity and apparent dissociation constants of 210 and 16.6 microM. AntR showed sub-millisecond Mn(II) on-rates as measured using stopped-flow EPR. The kinetics of Mn(II) dissociation, measured by displacement with Zn(II), was biphasic with rate constants of 35.7 and 0.115 s(-1). Variable-temperature parallel and perpendicular mode cw EPR spectra showed no evidence of a spin-exchange interaction, suggesting that the two Mn(II) ions are not forming a binuclear cluster. Finally, size exclusion chromatography and double electron-electron resonance EPR demonstrated that AntR forms a dimer in the absence of Mn(II). These results provide insights into the metal activation of AntR and allow a comparison with related DtxR proteins.
Asunto(s)
Bacillus anthracis/química , Manganeso/metabolismo , Proteínas Represoras/metabolismo , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Represoras/químicaRESUMEN
The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is the prototypic member of a superfamily of transition metal ion-activated transcriptional regulators that have been isolated from Gram-positive prokaryotes. Upon binding divalent transition metal ions, the N-terminal domain of DtxR undergoes a dynamic structural organization leading to homodimerization and target DNA binding. We have used site-directed mutagenesis and NMR analysis to probe the mechanism by which apo-DtxR transits from an inactive to a fully active repressor upon metal ion binding. We demonstrate that the ancillary metal-binding site mutant DtxR(H79A) requires higher concentrations of metal ions for activation both in vivo and in vitro, providing a functional correlation to the proposed cooperativity between ancillary and primary binding sites. We also demonstrate that the C-terminal src homology 3 (SH3)-like domain of DtxR functions to modulate repressor activity by (i) binding to the polyprolyl tether region between the N- and C-terminal domains, and (ii) destabilizing the ancillary binding site, leading to full inactivation of the repressor. Finally, we show by NMR analysis that the hyperactive phenotype of DtxR(E175K) results from the stabilization of a structural intermediate in the activation process. Taken together, the data presented support a multistep model for the activation of apo-DtxR by transition metal ions.
Asunto(s)
Proteínas Bacterianas/genética , Corynebacterium diphtheriae/genética , Proteínas de Unión al ADN/genética , Alanina , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Proteínas de Unión al ADN/química , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Biosíntesis de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Transcripción Genética , beta-Galactosidasa/metabolismoRESUMEN
Spinal muscular atrophy (SMA) is a degenerative disorder of spinal motor neurons caused by homozygous mutations in the survival motor neuron (SMN1) gene. Because increased tissue levels of human SMN protein (hSMN) in transgenic mice reduce the motor neuron loss caused by murine SMN knockout, we engineered a recombinant SMN fusion protein to deliver exogenous hSMN to the cytosolic compartment of motor neurons. The fusion protein, SDT, is comprised of hSMN linked to the catalytic and transmembrane domains of diphtheria toxin (DTx) followed by fragment C of tetanus toxin (TTC). Following overexpression in Escherichia coli, SDT possessed a subunit molecular weight of approximately 130 kDa as revealed by both SDS-PAGE and immunoblot analyses with anti-SMN, anti-DTx, and anti-TTC antibodies. Like wild-type SMN, purified SDT showed specific binding in vitro to an RG peptide derived from Ewing's sarcoma protein. The fusion protein also bound to cultured primary neurons in amounts similar to those achieved by TTC. Unlike the case with TTC, however, immunolabeling of SDT-treated neurons with anti-TTC and anti-SMN antibodies showed staining restricted to the cell surface. Results from cytotoxicity studies in which the DTx catalytic domain of SDT was used as a reporter protein for internalization and membrane translocation activity suggest that the SMN moiety of the fusion protein is interfering with one or both of these processes. While these studies indicate that SDT may not be useful for SMA therapy, the use of the TTC:DTx fusion construct to deliver other passenger proteins to the neuronal cytosol should not be ruled out.
Asunto(s)
Atrofia Muscular Espinal/tratamiento farmacológico , Proteínas del Tejido Nervioso/genética , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/farmacología , Toxina Tetánica/genética , Animales , Animales Recién Nacidos , Anticuerpos/inmunología , Membrana Celular/efectos de los fármacos , Membrana Celular/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Citotoxinas/genética , Citotoxinas/inmunología , Citotoxinas/farmacología , Toxina Diftérica/genética , Toxina Diftérica/inmunología , Relación Dosis-Respuesta a Droga , Endocitosis/inmunología , Inmunohistoquímica , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteínas del Tejido Nervioso/inmunología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fragmentos de Péptidos/inmunología , Unión Proteica/inmunología , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/inmunología , Proteínas de Unión al ARN , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas del Complejo SMN , Proteína 1 para la Supervivencia de la Neurona Motora , Toxina Tetánica/inmunologíaRESUMEN
In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.
Asunto(s)
Dominio Catalítico , Citosol/metabolismo , Toxina Diftérica/metabolismo , Linfocitos T/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Benzoquinonas , Línea Celular , Endocitosis , Endosomas/metabolismo , Proteínas HSP90 de Choque Térmico/análisis , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas , Lactonas/farmacología , Macrólidos , Espectrometría de Masas , Factor 2 de Elongación Peptídica/metabolismo , Quinonas/farmacología , Reductasa de Tiorredoxina-Disulfuro/análisis , Reductasa de Tiorredoxina-Disulfuro/efectos de los fármacos , LevadurasRESUMEN
The diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor that acts as a global regulatory element in the control of iron-sensitive genes in Corynebacterium diphtheriae. We recently described (L. Sun, J. C. vanderSpek, and J. R. Murphy, Proc. Natl. Acad. Sci. USA 95:14985-14990, 1998) the isolation and in vivo characterization of a hyperactive mutant of DtxR, DtxR(E175K), that appeared to be constitutively active. We demonstrate here that while DtxR(E175K) remains active in vivo in the presence of 300 micro M 2,2'dipyridyl, the purified repressor is, in fact, dependent upon low levels of transition metal ion to transit from the inactive apo form to the active metal ion-bound form of the repressor. Binding studies using 8-anilino-1-naphthalenesulfonic acid suggest that the E175K mutation stabilizes an intermediate of the molten-globule form of the repressor, increasing exposure of hydrophobic residues to solvent. We demonstrate that the hyperactive DtxR(E175K) phenotype is dependent upon an intact ancillary metal ion-binding site (site 1) of the repressor. These observations support the hypothesis that metal ion binding in the ancillary site facilitates the conversion of the inactive apo-repressor to its active, operator-binding conformation. Furthermore, these results support the hypothesis that the C-terminal src homology 3-like domain of DtxR plays an active role in the modulation of repressor activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Metales/metabolismo , Proteínas Represoras/metabolismo , Sustitución de Aminoácidos , Naftalenosulfonatos de Anilina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Cationes/metabolismo , Corynebacterium diphtheriae/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Colorantes Fluorescentes/metabolismo , Regulación Bacteriana de la Expresión Génica , Mutación , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Dominios Homologos srcRESUMEN
The residues located at the carboxyl terminus of helix D in interleukin-7 (IL-7) have previously been targeted as important for recruitment and binding to the gamma chain component of the IL-7 receptor (IL-7R). In this study, Trp 143 of helix D was mutated to His, Phe, Tyr and Pro and these mutants, along with a W143A mutant previously described, were studied to determine the effects on activation of DNA synthesis and binding affinity to IL-7R positive 2E8 cells. The W143F and W143Y mutants were similar to wild type IL-7 in their binding properties and retained 85% and 74% of their activating properties, respectively. In contrast, the W143H mutant possessed a lower binding affinity and a corresponding decrease in activation, the W143A mutant possessed an over 100-fold decreased binding affinity and some residual activation activity and the W143P mutant possessed a greatly decreased binding affinity and did not activate. These results strongly suggest an aromatic residue is required at position 143 for IL-7R binding and subsequent signal transduction.