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1.
J Mol Biol ; 432(14): 4092-4107, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32439331

RESUMO

Transcription elongation factor Spt6 associates with RNA polymerase II (RNAP II) via a tandem SH2 (tSH2) domain. The mechanism and significance of the RNAP II-Spt6 interaction is still unclear. Recently, it was proposed that Spt6-tSH2 is recruited via a newly described phosphorylated linker between the Rpb1 core and its C-terminal domain (CTD). Here, we report binding studies with isolated tSH2 of Spt6 (Spt6-tSH2) and Spt6 lacking the first unstructured 297 residues (Spt6ΔN) with a minimal CTD substrate of two repetitive heptads phosphorylated at different sites. The data demonstrate that Spt6 also binds the phosphorylated CTD, a site that was originally proposed as a recognition epitope. We also show that an extended CTD substrate harboring 13 repetitive heptads of the tyrosine-phosphorylated CTD binds Spt6-tSH2 and Spt6ΔN with tighter affinity than the minimal CTD substrate. The enhanced binding is achieved by avidity originating from multiple phosphorylation marks present in the CTD. Interestingly, we found that the steric effects of additional domains in the Spt6ΔN construct partially obscure the binding of the tSH2 domain to the multivalent ligand. We show that Spt6-tSH2 binds various phosphorylation patterns in the CTD and found that the studied combinations of phospho-CTD marks (1,2; 1,5; 2,4; and 2,7) all facilitate the interaction of CTD with Spt6. Our structural studies reveal a plasticity of the tSH2 binding pockets that enables the accommodation of CTDs with phosphorylation marks in different registers.

2.
Nucleic Acids Res ; 48(4): 2091-2106, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31875226

RESUMO

Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3'UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. Our structure of the ARF1 SBS-STAU1 complex uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix α1, the ß1-ß2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix α2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitro binding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.


Assuntos
Fator 1 de Ribosilação do ADP/química , Proteínas do Citoesqueleto/química , Motivo de Ligação ao RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/química , Proteínas de Ligação a RNA/química , Fator 1 de Ribosilação do ADP/genética , Sítios de Ligação/genética , Citoplasma/química , Citoplasma/genética , Proteínas do Citoesqueleto/genética , Humanos , Conformação Proteica , Estabilidade de RNA/genética , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/genética
3.
Mol Cell Proteomics ; 18(2): 320-337, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30459217

RESUMO

Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. Structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40, as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo, supporting its functional role in vivo As human cytosolic Hsp70 can interact with tetratricopeptide repeat (TPR) domain containing cochaperones, we tested the interaction of Hsp70 ATP-dependent dimers with Chip and Tomm34 cochaperones. Although Chip associates with intact Hsp70 dimers to form a larger complex, binding of Tomm34 disrupts the Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests a novel role of TPR domain cochaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Espalhamento a Baixo Ângulo , Estresse Fisiológico
4.
Proc Natl Acad Sci U S A ; 114(42): 11133-11138, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29073019

RESUMO

RNA polymerase II contains a long C-terminal domain (CTD) that regulates interactions at the site of transcription. The CTD architecture remains poorly understood due to its low sequence complexity, dynamic phosphorylation patterns, and structural variability. We used integrative structural biology to visualize the architecture of the CTD in complex with Rtt103, a 3'-end RNA-processing and transcription termination factor. Rtt103 forms homodimers via its long coiled-coil domain and associates densely on the repetitive sequence of the phosphorylated CTD via its N-terminal CTD-interacting domain. The CTD-Rtt103 association opens the compact random coil structure of the CTD, leading to a beads-on-a-string topology in which the long rod-shaped Rtt103 dimers define the topological and mobility restraints of the entire assembly. These findings underpin the importance of the structural plasticity of the CTD, which is templated by a particular set of CTD-binding proteins.


Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química
5.
Nucleic Acids Res ; 45(21): 12170-12180, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28981702

RESUMO

Telomeric repeat binding factor 2 (TRF2) folds human telomeres into loops to prevent unwanted DNA repair and chromosome end-joining. The N-terminal basic domain of TRF2 (B-domain) protects the telomeric displacement loop (D-loop) from cleavage by endonucleases. Repressor activator protein 1 (Rap1) binds TRF2 and improves telomeric DNA recognition. We found that the B-domain of TRF2 stabilized the D-loop and thus reduced unwinding by BLM and RPA, whereas the formation of the Rap1-TRF2 complex restored DNA unwinding. To understand how the B-domain of TRF2 affects DNA binding and D-loop processing, we analyzed DNA binding of full-length TRF2 and a truncated TRF2 construct lacking the B-domain. We quantified how the B-domain improves TRF2's interaction with DNA via enhanced long-range electrostatic interactions. We developed a structural envelope model of the B-domain bound on DNA. The model revealed that the B-domain is flexible in solution but becomes rigid upon binding to telomeric DNA. We proposed a mechanism for how the B-domain stabilizes the D-loop.


Assuntos
DNA/química , Proteínas de Ligação a Telômeros/metabolismo , Telômero/química , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , DNA/metabolismo , Humanos , Ligação Proteica , Domínios Proteicos , Eletricidade Estática , Telômero/metabolismo
7.
J Biol Chem ; 292(42): 17525-17540, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28860196

RESUMO

Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the ß3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the ß3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the ß3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the ß3-α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic ß3-α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Proteínas Quinases/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cristalografia por Raios X , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Proteínas Quinases/genética , Estrutura Secundária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética
8.
Microbiol Res ; 188-189: 9-22, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27296958

RESUMO

UNLABELLED: The Pden_2689 gene encoding FerA, an NADH:flavin oxidoreductase required for growth of Paracoccus denitrificans under iron limitation, was cloned and overexpressed as a C-terminally His6-tagged derivative. The binding of substrates and products was detected and quantified by isothermal titration calorimetry and fluorometric titration. FerA binds FMN and FAD with comparable affinity in an enthalpically driven, entropically opposed process. The reduced flavin is bound more loosely than the oxidized one, which was confirmed by a negative shift in the redox potential of FMN after addition of FerA. Initial velocity and substrate analogs inhibition studies showed that FerA follows a random-ordered sequence of substrate (NADH and FMN) binding. The primary kinetic isotope effects from stereospecifically deuterated nicotinamide nucleotides demonstrated that hydride transfer occurs from the pro-S position and contributes to rate limitation for the overall reaction. The crystal structure of FerA revealed a twisted seven-stranded antiparallel ß-barrel similar to that of other short chain flavin reductases. Only minor structural changes around Arg106 took place upon FMN binding. The solution structure FerA derived from small angle X-ray scattering (SAXS) matched the dimer assembly predicted from the crystal structure. Site-directed mutagenesis pinpointed a role of Arg106 and His146 in binding of flavin and NADH, respectively. Pull down experiments performed with cytoplasmic extracts resulted in a negative outcome indicating that FerA might physiologically act without association with other proteins. Rapid kinetics experiments provided evidence for a stabilizing effect of another P. denitrificans protein, the NAD(P)H: acceptor oxidoreducase FerB, against spontaneous oxidation of the FerA-produced dihydroflavin.


Assuntos
FMN Redutase/química , FMN Redutase/metabolismo , Paracoccus denitrificans/enzimologia , Cromatografia de Afinidade , Clonagem Molecular , Cristalografia por Raios X , FMN Redutase/genética , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , NAD/metabolismo , Paracoccus denitrificans/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Espalhamento a Baixo Ângulo
9.
Chemistry ; 21(18): 6695-9, 2015 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-25765709

RESUMO

Herein, we report on the rational design, preparation and characterization of a novel homo-octanuclear helicate, which results from a spatial extension of the central tetranuclear platform. The 3D supramolecular assembly is obtained by complexing europium(III) with a new hexatopic tripodal ligand. The isolated octanuclear helicate is fully characterized by different methods clearly evidencing the structure predicted with molecular modelling. The ligand preorganization plays a crucial role in a successful self-assembly process and induces the formation of a well-defined triple-stranded helical structure. This prototypal octanuclear edifice accommodating functional lanthanides within a 3D scaffold offers attractive perspectives for further applications.

10.
PLoS One ; 9(5): e96262, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24817153

RESUMO

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.


Assuntos
Proteínas de Bactérias/química , NADH NADPH Oxirredutases/química , Paracoccus denitrificans/enzimologia , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Biocatálise , Domínio Catalítico/genética , Cristalografia por Raios X , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Flavinas/química , Flavinas/metabolismo , Flavoproteínas/química , Flavoproteínas/genética , Flavoproteínas/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NADH NADPH Oxirredutases/classificação , NADH NADPH Oxirredutases/metabolismo , NADP/química , NADP/metabolismo , Oxirredução , Paracoccus denitrificans/genética , Ligação Proteica , Multimerização Proteica , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difração de Raios X
11.
Plant J ; 67(5): 827-39, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21569135

RESUMO

Multistep phosphorelay (MSP) signaling mediates responses to a variety of important stimuli in plants. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-AHP5) to nuclear response regulators. In contrast to ancestral two-component signaling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. Here, we show that the C-terminal receiver domain of HK CKI1 (CKI1(RD) ) is responsible for the recognition of CKI1 downstream signaling partners, and specifically interacts with AHP2, AHP3 and AHP5 with different affinities. We studied the effects of Mg²âº, the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF3⁻ on CKI1(RD) in solution, and determined the crystal structure of free CKI1(RD) and CKI1(RD) in a complex with Mg²âº. We found that the structure of CKI1(RD) shares similarities with the only known structure of plant HK, ETR1(RD) , with the main differences being in loop L3. Magnesium binding induces the rearrangement of some residues around the active site of CKI1(RD) , as was determined by both X-ray crystallography and NMR spectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Fosfotransferases/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Cristalografia por Raios X , Histidina/metabolismo , Modelos Moleculares , Mutação , Fosforilação , Fosfotransferases/genética , Mapeamento de Interação de Proteínas , Proteínas Quinases/genética , Proteínas Quinases/isolamento & purificação , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão , Sensibilidade e Especificidade , Transdução de Sinais/fisiologia
12.
Artigo em Inglês | MEDLINE | ID: mdl-20383015

RESUMO

The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and most notably quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors NADH or NADPH. Recombinant unmodified and SeMet-substituted FerB were crystallized under similar conditions by the hanging-drop vapour-diffusion method with microseeding using PEG 4000 as the precipitant. FerB crystallized in several different crystal forms, some of which diffracted to approximately 1.8 A resolution. The crystals of native FerB belonged to space group P2(1), with unit-cell parameters a = 61.6, b = 110.1, c = 65.2 A, beta = 118.2 degrees and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5 A and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress.


Assuntos
NADH Desidrogenase/química , Paracoccus denitrificans/enzimologia , Cristalização , Cristalografia por Raios X , Microbiologia do Solo
13.
Artigo em Inglês | MEDLINE | ID: mdl-19407381

RESUMO

The receiver domain (RD) of a sensor histidine kinase (HK) catalyses the transphosphorylation reaction during the action of HKs in hormonal and abiotic signalling in plants. Crystals of the recombinant RD of the Arabidopsis thaliana HK CYTOKININ-INDEPENDENT1 (CKI1(RD)) have been obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant and glycerol as a cryoprotectant. The crystals diffracted to approximately 2.4 A resolution on beamline BW7B of the DORIS-III storage ring. The diffraction improved significantly after the use of a non-aqueous cryoprotectant. Crystals soaked in Paratone-N diffracted to at least 2.0 A resolution on beamline BW7B and their mosaicity decreased more than tenfold. The crystals belonged to space group C222(1), with unit-cell parameters a = 54.46, b = 99.82, c = 79.94 A. Assuming the presence of one molecule of the protein in the asymmetric unit gives a Matthews coefficient V(M) of 2.33 A(3) Da(-1). A molecular-replacement solution has been obtained and structure refinement is in progress.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Proteínas Quinases/química , Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Histidina Quinase , Proteínas Quinases/isolamento & purificação , Proteínas Quinases/metabolismo
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