RESUMEN
The redox properties of metallo-base pairs remain to be elucidated. Herein, we report the detailed 1H/13C/109Ag NMR spectroscopic and cyclic voltammetric characterisation of the [Ag(cytidine)2]+ complex as isolated cytosine-Ag+-cytosine (C-Ag+-C) base pairs. We also performed comparative studies between cytidine/Ag+ and other nucleoside/Ag+ systems by using cyclic voltammetry measurements. In addition, to evaluate the effect of [Ag(cytidine)2]+ formation on the chemical reduction of Ag+ to Ag, we utilised the redox reaction between Ag+ and tetrathiafulvalene (TTF). We found that Ag+-mediated base pairing lowers the redox potential of the Ag+/Ag couple. In addition, C-Ag+-C base pairing makes it more difficult to reduce captured Ag+ ions than in other nucleoside/Ag+ systems. Remarkably, the cytidine/Ag+ system can be utilised to control the redox potential of the Ag+/Ag couple in DMSO. This feature of the cytidine/Ag+ system may be exploited for Ag nanoparticle synthesis by using the redox reaction between Ag+ and TTF.
RESUMEN
Negative regulator differentiation 1 (Nrd1), a fission yeast RNA binding protein, modulates cytokinesis and sexual development and contributes to stress granule formation in response to environmental stresses. Nrd1 comprises four RRM domains and binds and stabilizes Cdc4 mRNA that encodes the myosin II light chain. Nrd1 binds the Cpc2 fission-yeast RACK1 homolog, and the interaction promotes Nrd1 localization to stress granules. Interestingly, Pmk1 mitogen-activated protein kinase phosphorylates Thr40 in the unstructured N-terminal region and Thr126 in the first RRM domain of Nrd1. Phosphorylation significantly reduces RNA-binding activity and likely modulates Nrd1 function. To reveal the relationship between the structure and function of Nrd1 and how phosphorylation affects structure, we used heteronuclear NMR techniques to investigate the three-dimensional structure of Nrd1. Here we report the 1H, 13C, and 15N resonance assignments of RRM1-RRM2 (residues 108-284) comprising the first and second RRMs obtained using heteronuclear NMR techniques. Secondary structures derived from the chemical shifts are reported. These data should contribute to the understanding of the three-dimensional structure of the RRM1-RRM2 region of Nrd1 and the perturbation caused by phosphorylation.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Ribonucleoproteínas/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces , Estructura Secundaria de Proteína , Ribonucleoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1â mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.
Asunto(s)
Bombyx/genética , Bombyx/metabolismo , Larva/metabolismo , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Proteínas Recombinantes/biosíntesis , Animales , Humanos , Larva/genética , Fosforilación , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes/genética , Transducción de SeñalRESUMEN
The transcriptional corepressors SMRT/NCoR, components of histone deacetylase complexes, interact with nuclear receptors and many other transcription factors. SMRT is a target for the ubiquitously expressed protein kinase CK2, which is known to phosphorylate a wide variety of substrates. Increasing evidence suggests that CK2 plays a regulatory role in many cellular events, particularly, in transcription. However, little is known about the precise mode of action involved. Here, we report the three-dimensional structure of a SMRT/HDAC1-associated repressor protein (SHARP) in complex with phosphorylated SMRT, as determined by solution NMR. Phosphorylation of the CK2 site on SMRT significantly increased affinity for SHARP. We also confirmed the significance of CK2 phosphorylation by reporter assay and propose a mechanism involving the process of phosphorylation acting as a molecular switch. Finally, we propose that the SPOC domain functions as a phosphorylation binding module.
Asunto(s)
Quinasa de la Caseína II/química , Proteínas de Homeodominio/química , Proteínas Nucleares/química , Co-Represor 2 de Receptor Nuclear/química , Sitios de Unión , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Proteínas de Unión al ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Co-Represor 2 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/metabolismo , Fosforilación , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Sitios de Unión , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Soluciones , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.
Asunto(s)
Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Co-Represor 2 de Receptor Nuclear/química , Péptidos/química , Secuencia de Aminoácidos , Proteínas de Unión al ADN , Humanos , Co-Represor 2 de Receptor Nuclear/metabolismo , Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas de Unión al ARNRESUMEN
End-binding protein 1 (EB1) is one of the best studied plus-end tracking proteins. It is known that EB1 specifically binds the plus ends of microtubules (MTs) and promotes MT growth. EB1 activity is thought to be autoinhibited by an intramolecular interaction. Recent cryo-EM analyses showed that the CH domain of Mal3p (Schizosaccharomyces pombe EB1 homolog) binds to GMPCPP-MT (Sandblad, L. Cell 127 (2006) 1415-24), and strongly binds GTPγS-MT which is proposed to mimic MT plus ends better than GMPCPP-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Here, we report on the MT binding sites of the CH domain of EB1 as revealed by NMR using the transferred cross-saturation method. In this study, we used GMPCPP-MT and found that the MT binding sites are very similar to the binding site for GTPγS-MT as suggested by cryo-EM (Maurer S.P. et al. Cell 149 (2012) 371-82). Notably, the N-terminal tip of helix α6 of the CH domain did not make contact with GMPCPP-MT, in contrast to the cryo-EM study which showed that it is closely located to a putative switch region of ß-tubulin in GTPγS-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Further, we found that the intramolecular interaction site of EB1 overlaps the MT binding sites, indicating that the MT binding sites are masked by interaction with the C-terminal domain. We propose a structural view of autoinhibition and its release mechanism through competition binding with binding partners such as adenomatous polyposis coli protein.
Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Modelos Moleculares , Proteína de la Poliposis Adenomatosa del Colon/química , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Escherichia coli Orf135 hydrolyzes oxidatively damaged nucleotides such as 2-hydroxy-dATP, 8-oxo-dGTP and 5-hydroxy-CTP, in addition to 5-methyl-dCTP, dCTP and CTP. Nucleotide pool sanitization by Orf135 is important since nucleotides are continually subjected to potential damage by reactive oxygen species produced during respiration. Orf135 is a member of the Nudix family of proteins which hydrolyze nucleoside diphosphate derivatives. Nudix hydrolases are characterized by the presence of a conserved motif, even though they recognize various substrates and possess a variety of substrate binding pockets. We investigated the tertiary structure of Orf135 and its interaction with a 2-hydroxy-dATP analog using NMR. We report on the solution structure of Orf135, which should contribute towards a structural understanding of Orf135 and its interaction with substrates.