RESUMO
Mutations in PARK7, the gene encoding the DJ-1 protein, are associated with early onset of Parkinson's disease. The C106 residue of DJ-1 is highly susceptible to oxidation, and its oxidation status is essential for various in vivo neuroprotective roles. Since C106 is readily oxidized to sulfinic acid that is not reduced by dithiothreitol, no method to separate native DJ-1 protein from the oxidized one creates challenges in the in vitro study of the biological relevance of C106-oxidation state. Here, we report an efficient column chromatography method to purify native, C106-sulfinic, and mixed (combination of the priors) forms of DJ-1. This method will be useful for systematic in vitro studies of DJ-1 functions by providing specific native and C106-sulfinic DJ-1 proteins.
Assuntos
Proteínas Oncogênicas , Doença de Parkinson , Cromatografia , Humanos , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Oxirredução , Estresse Oxidativo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismoRESUMO
Distal-less 3 (Dlx3) is a homeobox-containing transcription factor and plays a crucial role in the development and differentiation process. Human Dlx3 consists of two transactivation domains and a homeobox domain (HD) that selectively binds to the consensus site (5'-TAATT-3') of the DNA duplex. Here, we performed chemical shift perturbation experiments on Dlx3-HD in a complex with a 10-base-paired (10-bp) DNA duplex under various salt conditions. We also acquired the imino proton spectra of the 10-bp DNA to monitor the changes in base-pair stabilities during titration with Dlx3-HD. Our study demonstrates that Dlx3-HD selectively recognizes its consensus DNA sequences through the α3 helix and L1 loop regions with a unique dynamic feature. The dynamic properties of the binding of Dlx3-HD to its consensus DNA sequence can be modulated by varying the salt concentrations. Our study suggested that this unique structural and dynamic feature of Dlx3-HD plays an important role in target DNA recognition, which might be associated with tricho-dento-osseous syndrome.
Assuntos
Proteínas de Homeodomínio , Sais , Fatores de Transcrição , DNA/metabolismo , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Sais/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The BRG1-associated factor 60A (BAF60A), an SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1, has been known to be important for transcriptional activation and inhibition through the alteration of the DNA nucleosome. Although the association between BAF60A and p53 plays a critical role in tumor suppression, the interaction mode is still unclear. Here, we report the detailed interactions between BAF60A and p53 by both NMR spectroscopy and pull-down analysis. Both N-terminal region (BAF60ANR) and the SWIB domain (BAF60ASWIB) of BAF60A directly interact with the tetramerization domain of p53 (p53TET). NMR data show that Ile315, Met366, Ala388, and Tyr390 of BAF60ASWIB are mostly involved in p53TET binding. The calculated dissociation constant (KD) value between BAF60ASWIB and p53TET revealed relatively weak binding affinity, at approximately 0.3 ± 0.065 mM. Our data will enhance detailed interaction mechanism to elucidate the molecular basis of p53-mediated integration via BAF60A interaction.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Humanos , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteína Supressora de Tumor p53/genéticaRESUMO
Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in µs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZαADAR1)-DNA complex. The global search results indicated that a α-helical segment of hZαADAR1 provided the main contribution to the three-state conformational changes of a hZαADAR1-DNA complex with a slow B-Z exchange process. The two global exchange rate constants, kex and kZB, were found to be 844 and 9.8 s-1, respectively, in agreement with two regimes of residue-dependent chemical shift differences-the "dominant oscillatory regime" and "semi-oscillatory regime". We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.
Assuntos
Adenosina Desaminase/química , DNA de Forma B/química , DNA Forma Z/química , Modelos Moleculares , Proteínas de Ligação a RNA/química , Adenosina Desaminase/metabolismo , Algoritmos , DNA de Forma B/metabolismo , DNA Forma Z/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Conformação Proteica , Proteínas de Ligação a RNA/metabolismo , TermodinâmicaRESUMO
Monoclonal antibody (mAb) drugs are clinically important for the treatment of various diseases. TTAC-0001 is under development as a new anti-cancer antibody drug targeting VEGFR-2. As the less severe toxicity of TTAC-0001 compared to Bevacizumab, likely due to the decreased in vivo half-life, seems to be related to its structural flexibility, it is important to map the exact flexible regions. Although the 13C/15N-labeled protein is required for NMR analyses, it is difficult to obtain antibody fragments (Fab and scFv) containing disulfide bonds through general cytosolic expression in Escherichia coli (E. coli). Here, we notably increased the periplasmic expression of the 13C/15N-labeled TTAC-0001-Fab (13C/15N-TTAC-Fab) through simple isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction at an increased optical density (1.5 OD600nm). Through NMR triple resonance experiments, two loop insertions (LI-1 between the VH and CH1; LI-2 between the VL and CL) were confirmed to be highly flexible. The additional LIs could be another way to engineer the antibody by changing the pharmacokinetic properties.
Assuntos
Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Anticorpos Monoclonais Humanizados/genética , Isótopos de Carbono , Dissulfetos/química , Escherichia coli/genética , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Isopropiltiogalactosídeo , Marcação por Isótopo , Isótopos de Nitrogênio , Periplasma/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Neuraminidase (NA), one of the major surface glycoproteins of influenza A virus (IAV), is an important diagnostic biomarker and antiviral therapeutic target. Cytolysin A (ClyA) is a nanopore sensor with an internal constriction of 3.3 nm, enabling the detection of protein conformations at the single-molecule level. In this study, a nanopore-based approach is developed for analysis of the enzymatic activity of NA, which facilitates rapid and highly sensitive diagnosis of IAV. Current blockade analysis of the d-glucose/d-galactose-binding protein (GBP) trapped within a type I ClyA-AS (ClyA mutant) nanopore reveals that galactose cleaved from sialyl-galactose by NA of the influenza virus can be detected in real time and at the single-molecule level. Our results show that this nanopore sensor can quantitatively measure the activity of NA with 40-80-fold higher sensitivity than those previously reported. Furthermore, the inhibition of NA is monitored using small-molecule antiviral drugs, such as zanamivir. Taken together, our results reveal that the ClyA protein nanopore can be a valuable platform for the rapid and sensitive point-of-care diagnosis of influenza and for drug screening against the NA target.
Assuntos
Citotoxinas/metabolismo , Ensaios Enzimáticos/métodos , Vírus da Influenza A/enzimologia , Nanoporos , Neuraminidase/metabolismo , Citotoxinas/química , Modelos Moleculares , Neuraminidase/química , Conformação ProteicaRESUMO
Human SNF5 and BAF155 constitute the core subunit of multi-protein SWI/SNF chromatin-remodeling complexes that are required for ATP-dependent nucleosome mobility and transcriptional control. Human SNF5 (hSNF5) utilizes its repeat 1 (RPT1) domain to associate with the SWIRM domain of BAF155. Here, we employed X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and various biophysical methods in order to investigate the detailed binding mechanism between hSNF5 and BAF155. Multi-angle light scattering data clearly indicate that hSNF5171-258 and BAF155SWIRM are both monomeric in solution and they form a heterodimer. NMR data and crystal structure of the hSNF5171-258/BAF155SWIRM complex further reveal a unique binding interface, which involves a coil-to-helix transition upon protein binding. The newly formed αN helix of hSNF5171-258 interacts with the ß2-α1 loop of hSNF5 via hydrogen bonds and it also displays a hydrophobic interaction with BAF155SWIRM. Therefore, the N-terminal region of hSNF5171-258 plays an important role in tumorigenesis and our data will provide a structural clue for the pathogenesis of Rhabdoid tumors and malignant melanomas that originate from mutations in the N-terminal loop region of hSNF5.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Mutação , Nucleossomos/genética , Proteína SMARCB1/genética , Fatores de Transcrição/genética , Sítios de Ligação/genética , Dicroísmo Circular , Cristalografia por Raios X , Regulação da Expressão Gênica , Humanos , Espectroscopia de Ressonância Magnética , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Nucleossomos/metabolismo , Ligação Proteica , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patologia , Proteína SMARCB1/química , Proteína SMARCB1/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Mitochondrial E3 ubiquitin ligase 1 (MUL1) is a multifunctional mitochondrial protein involved in various biological processes such as mitochondrial dynamics, cell growth, apoptosis, and mitophagy. MUL1 mediates the ubiquitylation of mitochondrial p53 for proteasomal degradation. Although the interaction of MUL1-RING domain with its substrate, p53, is a unique mechanism in RING-mediated ubiquitylation, the molecular basis of this process remains unknown. In this study, we determined the solution structure of the MUL1-RING domain and characterized its interaction with the p53 transactivation domain (p53-TAD) by nuclear magnetic resonance (NMR) spectroscopy. The overall structure of the MUL1-RING domain is similar to those of RING domains of other E3 ubiquitinases. The MUL1-RING domain adopts a ßßαß fold with three anti-parallel ß-strands and one α-helix, containing a canonical cross-brace motif for the ligation of two zinc ions. Through NMR chemical shift perturbation experiments, we determined the p53-TAD-binding site in the MUL1-RING domain and showed that the MUL1-RING domain interacts mainly with the p53-TAD2 subdomain composed of residues 39-57. Taken together, our results provide a molecular basis for the novel recognition mechanism of the p53-TAD substrate by the MUL1-RING domain.
Assuntos
Espectroscopia de Ressonância Magnética , Domínios RING Finger , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Humanos , Ligação Proteica , Especificidade por Substrato , UbiquitinaçãoRESUMO
Cold-shock proteins (Csps) are expressed at lower-than-optimum temperatures, and they function as RNA chaperones; however, no structural studies on psychrophilic Csps have been reported. Here, we aimed to investigate the structure and dynamics of the Csp of psychrophile Colwellia psychrerythraea 34H, ( Cp-Csp). Although Cp-Csp shares sequence homology, common folding patterns, and motifs, including a five ß-stranded barrel, with its thermophilic counterparts, its thermostability (37 °C) was markedly lower than those of other Csps. Cp-Csp binds heptathymidine with an affinity of 10-7 M, thereby increasing its thermostability to 50 °C. Nuclear magnetic resonance spectroscopic analysis of the Cp-Csp structure and backbone dynamics revealed a flexible structure with only one salt bridge and 10 residues in the hydrophobic cavity. Notably, Cp-Csp contains Tyr51 instead of the conserved Phe in the hydrophobic core, and its phenolic hydroxyl group projects toward the surface. The Y51F mutation increased the stability of hydrophobic packing and may have allowed for the formation of a K3-E21 salt bridge, thereby increasing its thermostability to 43 °C. Cp-Csp exhibited conformational exchanges in its ribonucleoprotein motifs 1 and 2 (754 and 642 s-1), and heptathymidine binding markedly decreased these motions. Cp-Csp lacks salt bridges and has longer flexible loops and a less compact hydrophobic cavity resulting from Tyr51 compared to mesophilic and thermophilic Csps. These might explain the low thermostability of Cp-Csp. The conformational flexibility of Cp-Csp facilitates its accommodation of nucleic acids at low temperatures in polar oceans and its function as an RNA chaperone for cold adaptation.
Assuntos
Alteromonadaceae/química , Proteínas de Bactérias/química , Proteínas e Peptídeos de Choque Frio/química , Alteromonadaceae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas e Peptídeos de Choque Frio/metabolismo , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Alinhamento de Sequência , Timidina/análogos & derivados , Timidina/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMO
Quorum sensing (QS) regulates many natural phenotypes (e.q. virulence, biofilm formation, antibiotic resistance), and its components, when incorporated into synthetic genetic circuits, enable user-directed phenotypes. We created a library of Escherichia coli lsr operon promoters using error-prone PCR (ePCR) and selected for promoters that provided E. coli with higher tetracycline resistance over the native promoter when placed upstream of the tet(C) gene. Among the fourteen clones identified, we found several mutations in the binding sites of QS repressor, LsrR. Using site-directed mutagenesis we restored all p-lsrR-box sites to the native sequence in order to maintain LsrR repression of the promoter, preserving the other mutations for analysis. Two promoter variants, EP01rec and EP14rec, were discovered exhibiting enhanced protein expression. In turn, these variants retained their ability to exhibit the LsrR-mediated QS switching activity. Their sequences suggest regulatory linkage between CytR (CRP repressor) and LsrR. These promoters improve upon the native system and exhibit advantages over synthetic QS promoters previously reported. Incorporation of these promoters will facilitate future applications of QS-regulation in synthetic biology and metabolic engineering.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Evolução Molecular , Óperon , Percepção de Quorum/genética , Sequência de Bases , Sítios de Ligação , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutação , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Elementos de Resposta , Biologia Sintética , Transcrição GênicaRESUMO
Z-DNA binding proteins (ZBPs) play important roles in RNA editing, innate immune response and viral infection. Structural and biophysical studies show that ZBPs initially form an intermediate complex with B-DNA for B-Z conversion. However, a comprehensive understanding of the mechanism of Z-DNA binding and B-Z transition is still lacking, due to the absence of structural information on the intermediate complex. Here, we report the solution structure of the Zα domain of the ZBP-containing protein kinase from Carassius auratus(caZαPKZ). We quantitatively determined the binding affinity of caZαPKZ for both B-DNA and Z-DNA and characterized its B-Z transition activity, which is modulated by varying the salt concentration. Our results suggest that the intermediate complex formed by caZαPKZ and B-DNA can be used as molecular ruler, to measure the degree to which DNA transitions to the Z isoform.
Assuntos
DNA de Forma B/química , DNA Forma Z/química , Proteínas de Ligação a DNA/química , Proteínas de Peixes/química , Carpa Dourada/metabolismo , Proteínas Quinases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , DNA de Forma B/metabolismo , DNA Forma Z/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Cloreto de Sódio/química , TermodinâmicaRESUMO
A Z-DNA binding protein (ZBP)-containing protein kinase (PKZ) in fish species has an important role in the innate immune response. Previous structural studies of the Zα domain of the PKZ from Carassius auratus (caZαPKZ) showed that the protein initially binds to B-DNA and induces B-Z transition of double stranded DNA in a salt concentration-dependent manner. However, the significantly reduced B-Z transition activity of caZαPKZ at high salt concentration was not fully understood. In this study, we present the binding affinity of the protein for B-DNA and Z-DNA and characterize its extremely low B-Z transition activity at 250 mM NaCl. Our results emphasize that the B-DNA-bound form of caZαPKZ can be used as molecular ruler to measure the degree of B-Z transition.
Assuntos
DNA de Forma B/química , DNA Forma Z/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas Quinases/química , Proteínas Quinases/ultraestrutura , Cloreto de Sódio/química , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/ultraestrutura , Sítios de Ligação , DNA de Forma B/ultraestrutura , DNA Forma Z/ultraestrutura , Ativação Enzimática , Cinética , Ligação ProteicaRESUMO
S100A5 is a calcium-binding protein of S100 family, which represents a major ligand to the receptor for advanced glycation end product (RAGE), a pattern recognition receptor engaged in diverse pathological processes. Here we have characterized calcium binding of S100A5 and the complex formation between S100A5 and RAGE using calorimetry and NMR spectroscopy. S100A5 binds to calcium ions in a sequential manner with the equilibrium dissociation constants (KD) of 1.3 µM and 3.5 µM, which corresponds to the calcium-binding at the C-terminal and N-terminal EF-hands. Upon calcium binding, S100A5 interacts with the V domain of RAGE (RAGE-v) to form a heterotrimer (KD â¼5.9 µM) that is distinct among the S100 family proteins. Chemical shift perturbation data from NMR titration experiments indicates that S100A5 employs the periphery of the dimer interface to interact with RAGE-v. Distinct binding mode and stoichiometry of RAGE against different S100 family proteins could be important to modulate diverse RAGE signaling.
Assuntos
Antígenos de Neoplasias/metabolismo , Cálcio/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas S100/metabolismo , Calorimetria , Cromatografia , Motivos EF Hand , Escherichia coli/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , TermodinâmicaRESUMO
Peptide nanostructure has been widely explored for drug-delivery systems in recent studies. Peptides possess comparatively lower cytotoxicity and are more efficient than polymeric carriers. Here, we propose a peptide nanorod system, composed of an amphiphilic oligo-peptide RH3F8 (Arg-His3-Phe8), as a drug-delivery carrier. Arginine is an essential amino acid in typical cell-penetration peptides, and histidine induces endo- and lysosomal escape because of its proton sponge effect. Phenylalanine is introduced to provide rich hydrophobicity for stable self-assembly and drug encapsulation. The self-assembled structure of RH3F8 showed nanorod-shaped morphology, positive surface charge, and retained formation in water for 35 days. RH3F8, labeled with Nile Red, showed high cellar uptake and accumulation in both cytoplasm and nucleus. The RH3F8 nanorods demonstrated negligible cytotoxicity, as shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and hemolysis assays. To confirm the efficiency of drug delivery, curcumin was encapsulated in the RH3F8 nanorod system (RH3F8-Cur). RH3F8-Cur showed high encapsulation efficiency (24.63%) under the conditions of 200 µM curcumin. The RH3F8-Cur retained nanoscale size and positive surface charge, similar to those of the empty RH3F8 nanorods. RH3F8-Cur displayed a robust anticancer effect in HeLa and A549 cells, and inhibited the proliferation of cancer cells in a zebrafish model. These results indicate that the RH3F8 nanorods may be a promising candidate for a safe and effective drug-delivery system.
Assuntos
Antineoplásicos/administração & dosagem , Curcumina/administração & dosagem , Portadores de Fármacos/química , Nanotubos/química , Oligopeptídeos/química , Fenilalanina/análogos & derivados , Tensoativos/química , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/toxicidade , Linhagem Celular Tumoral , Curcumina/farmacologia , Portadores de Fármacos/metabolismo , Portadores de Fármacos/toxicidade , Células HeLa , Hemólise/efeitos dos fármacos , Humanos , Nanotubos/ultraestrutura , Neoplasias/tratamento farmacológico , Oligopeptídeos/metabolismo , Oligopeptídeos/toxicidade , Fenilalanina/metabolismo , Fenilalanina/toxicidade , Tensoativos/metabolismo , Tensoativos/toxicidade , Peixe-ZebraRESUMO
The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize Lys-48-linked polyubiquitins without an E3 ligase but how that is accomplished has been unclear. Although both E2s contain essential acidic loops, only Ube2r1 requires an additional C-terminal extension (184-196) for efficient Lys-48-ubiquitylation activity. The presence of Tyr-102 and Tyr-104 in the Ube2g1 acidic loop enhanced both ubiquitin binding and Lys-48-ubiquitylation and distinguished Ube2g1 from the otherwise similar truncated Ube2r1(1-183) (Ube2r1C). Replacement of Gln-105-Ser-106-Gly-107 in the acidic loop of Ube2r1C (Ube2r1C(YGY)) by the corresponding residues from Ube2g1 (Tyr-102-Gly-103-Tyr-104) increased Lys-48-ubiquitylation activity and ubiquitin binding. Two E2â¼UB thioester mimics (oxyester and disulfide) were prepared to characterize the ubiquitin binding activity of the acidic loop. The oxyester but not the disulfide derivative was found to be a functional equivalent of the E2â¼UB thioester. The ubiquitin moiety of the Ube2r1C(C93S)-[(15)N]UB(K48R) oxyester displayed two-state conformational exchange, whereas the Ube2r1C(C93S/YGY)-[(15)N]UB(K48R) oxyester showed predominantly one state. Together with NMR studies that compared UB(K48R) oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, in vitro ubiquitylation assays with various mutation forms of the E2s revealed how the intramolecular interaction between the acidic loop and the attached donor ubiquitin regulates Lys-48-ubiquitylation activity.
Assuntos
Lisina/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina/química , Sequência de Aminoácidos , Domínio Catalítico , Dissulfetos/química , Ésteres/química , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutação , Poliubiquitina/química , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more ß-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.
Assuntos
Escherichia coli K12/química , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico HSP30/química , Dobramento de Proteína , Sequência de Aminoácidos , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP30/metabolismo , Temperatura Alta , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
The kinetics of translocation of the homeodomain transcription factor HoxD9 between specific sites of the same or opposite polarities on the same DNA molecule have been studied by (15)Nz-exchange NMR spectroscopy. We show that exchange occurs by two facilitated diffusion mechanisms: a second-order intermolecular exchange reaction between specific sites located on different DNA molecules without the protein dissociating into free solution that predominates at high concentrations of free DNA, and a first-order intramolecular process involving direct transfer between specific sites located on the same DNA molecule. Control experiments using a mixture of two DNA molecules, each possessing only a single specific site, indicate that transfer between specific sites by full dissociation of HoxD9 into solution followed by reassociation is too slow to measure by z-exchange spectroscopy. Intramolecular transfer with comparable rate constants occurs between sites of the same and opposing polarity, indicating that both rotation-coupled sliding and hopping/flipping (analogous to geminate recombination) occur. The half-life for intramolecular transfer (0.5-1 s) is many orders of magnitude larger than the calculated transfer time (1-100 µs) by sliding, leading us to conclude that the intramolecular transfer rates measured by z-exchange spectroscopy represent the rate-limiting step for a one-base-pair shift from the specific site to the immediately adjacent nonspecific site. At zero concentration of added salt, the intramolecular transfer rate constants between sites of opposing polarity are smaller than those between sites of the same polarity, suggesting that hopping/flipping may become rate-limiting at very low salt concentrations.
Assuntos
DNA/química , Proteínas de Homeodomínio/química , Proteínas de Neoplasias/química , DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Cinética , Proteínas de Neoplasias/metabolismo , Isótopos de Nitrogênio , Ressonância Magnética Nuclear BiomolecularRESUMO
RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1-α2 loop and α3 region), and the aromatic side chain on the top of the ß-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , RecQ Helicases/química , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , SoluçõesRESUMO
Methylene blue (MB) has recently completed a Phase-3 clinical trial as leuco-methylthioninium (LMT) bis(hydromethanesulfonate) for treating Alzheimer's disease. Herein, we investigated the mechanism underlying the MB inhibition of tubulin-associated unit (tau) aggregation by focusing on tau monomers. We found that MB causes disulfide bond formation, resulting in strong nuclear magnetic resonance chemical shift perturbations in a large area of tau proteins. The oxidized form of MB, namely methylthioninium (MT+), specifically catalyzed the oxidation of cysteine residues in tau proteins to form disulfide bonds directly using O2. This process is independent of the MT+-to-LMT redox cycle. Moreover, MT+ preferentially oxidized C291 and C322 in the lysine-rich R2 and R3 domains. Under in vivo brain physoxia conditions, LMT may convert to MT+, possibly interfering with tau fibrillation via disulfide bond formation.
RESUMO
Transcription factors specifically bind to their consensus sequence motifs and regulate transcription efficiency. Transcription factors are also able to non-specifically contact the phosphate backbone of DNA through electrostatic interaction. The homeodomain of Meis1 TALE human transcription factor (Meis1-HD) recognizes its target DNA sequences via two DNA contact regions, the L1-α1 region and the α3 helix (specific binding mode). This study demonstrates that the non-specific binding mode of Meis1-HD is the energetically favored process during DNA binding, achieved by the interaction of the L1-α1 region with the phosphate backbone. An NMR dynamics study suggests that non-specific binding might set up an intermediate structure which can then rapidly and easily find the consensus region on a long section of genomic DNA in a facilitated binding process. Structural analysis using NMR and molecular dynamics shows that key structural distortions in the Meis1-HD-DNA complex are induced by various single nucleotide mutations in the consensus sequence, resulting in decreased DNA binding affinity. Collectively, our results elucidate the detailed molecular mechanism of how Meis1-HD recognizes single nucleotide mutations within its consensus sequence: (i) through the conformational features of the α3 helix; and (ii) by the dynamic features (rigid or flexible) of the L1 loop and the α3 helix. These findings enhance our understanding of how single nucleotide mutations in transcription factor consensus sequences lead to dysfunctional transcription and, ultimately, human disease.