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1.
J Phys Chem Lett ; 10(19): 5917-5922, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31509419

RESUMO

Nuclear magnetic relaxation provides invaluable quantitative site-specific information on the dynamics of complex systems. Determining dynamics on nanosecond time scales requires relaxation measurements at low magnetic fields incompatible with high-resolution NMR. Here, we use a two-field NMR spectrometer to measure carbon-13 transverse and longitudinal relaxation rates at a field as low as 0.33 T (proton Larmor frequency 14 MHz) in specifically labeled side chains of the protein ubiquitin. The use of radiofrequency pulses enhances the accuracy of measurements as compared to high-resolution relaxometry approaches, where the sample is moved in the stray field of the superconducting magnet. Importantly, we demonstrate that accurate measurements at a single low magnetic field provide enough information to characterize complex motions on low nanosecond time scales, which opens a new window for the determination of site-specific nanosecond motions in complex systems such as proteins.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Isótopos de Carbono , Cinética , Campos Magnéticos , Movimento (Física) , Prótons , Ubiquitina/química
2.
Chem Commun (Camb) ; 55(70): 10432-10435, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31408066

RESUMO

RNA represents an extremely promising and yet challenging therapeutic target. Here, we report the design of a series of C-nucleosides as original RNA binders. Some of them bind strongly and selectively to A-site prokaryotic ribosomal RNA.


Assuntos
Nucleosídeos/metabolismo , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Dicroísmo Circular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Nucleosídeos/química , RNA Ribossômico/química , Termodinâmica
3.
Phys Chem Chem Phys ; 21(35): 18850-18865, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31432055

RESUMO

Proton detected solid-state NMR under fast magic-angle-spinning (MAS) conditions is currently redefining the applications of solid-state NMR, in particular in structural biology. Understanding the contributions to the spectral linewidth is thereby of paramount importance. When disregarding the sample-dependent inhomogeneous contributions, the NMR proton linewidth is defined by homogeneous broadening, which has incoherent and coherent contributions. Understanding and disentangling these different contributions in multi-spin systems like proteins is still an open issue. The coherent contribution is mainly caused by the dipolar interaction under MAS and is determined by the molecular structure and the proton chemical shifts. Numerical simulation approaches based on numerically exact direct integration of the Liouville-von Neumann equation can give valuable information about the lineshape, but are limited to small spin systems (<12 spins). We present an alternative simulation method for the coherent contributions based on the rapid and partially analytic calculation of the second moments of large spin systems. We first validate the method on a simple system by predicting the 19F linewidth in CaF2 under MAS. We compare simulation results to experimental data for microcrystalline ubiquitin (deuterated 100% back-exchanged at 110 kHz and fully-protonated at 125 kHz). Our results quantitatively explain the observed linewidth per-residue basis for the vast majority of residues.


Assuntos
Simulação por Computador , Modelos Químicos , Proteínas/química , Ressonância Magnética Nuclear Biomolecular , Prótons
4.
Chem Commun (Camb) ; 55(75): 11215-11218, 2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31469130

RESUMO

Here we report the dephosphorylation and proteolysis of phosphorylated α-synuclein, a Parkinson's disease-related protein, in living cells in a time resolved manner using in-cell NMR.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Oócitos/metabolismo , alfa-Sinucleína/metabolismo , Animais , Oócitos/química , Fosforilação , Proteólise , Xenopus laevis , alfa-Sinucleína/química
5.
Nat Methods ; 16(8): 743-749, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31363225

RESUMO

Cellular behavior is controlled by the interplay of diverse biomolecules. Most experimental methods, however, can only monitor a single molecule class or reaction type at a time. We developed an in vitro nuclear magnetic resonance spectroscopy (NMR) approach, which permitted dynamic quantification of an entire 'heterotypic' network-simultaneously monitoring three distinct molecule classes (metabolites, proteins and RNA) and all elementary reaction types (bimolecular interactions, catalysis, unimolecular changes). Focusing on an eight-reaction co-transcriptional RNA folding network, in a single sample we recorded over 35 time points with over 170 observables each, and accurately determined five core reaction constants in multiplex. This reconstruction revealed unexpected cross-talk between the different reactions. We further observed dynamic phase-separation in a system of five distinct RNA-binding domains in the course of the RNA transcription reaction. Our Systems NMR approach provides a deeper understanding of biological network dynamics by combining the dynamic resolution of biochemical assays and the multiplexing ability of 'omics'.


Assuntos
Redes Reguladoras de Genes , Metaboloma , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/análise , RNA/análise , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Conformação Proteica , Proteínas/química , RNA/química , Dobramento de RNA
6.
Chemistry ; 25(50): 11635-11640, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31368214

RESUMO

Disulfide-containing detergents (DCDs) are introduced, which contain a disulfide bond in the hydrophobic tail. DCDs form smaller micelles than corresponding detergents with linear hydrocarbon chains, while providing good solubilization and reconstitution of membrane proteins. The use of this new class of detergents in structural biology is illustrated with solution NMR spectra of the human G protein-coupled receptor A2A AR, which is an α-helical protein, and the ß-barrel protein OmpX from E. coli.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Detergentes/química , Proteínas de Escherichia coli/química , Hidrolases/química , Receptor A2A de Adenosina/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Dissulfetos/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Hidrolases/metabolismo , Micelas , Ressonância Magnética Nuclear Biomolecular , Estabilidade Proteica , Receptor A2A de Adenosina/metabolismo
7.
J Phys Chem Lett ; 10(16): 4720-4724, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31369281

RESUMO

Probing the atomic details of intrinsically disordered proteins is crucial to understanding their biological function and relation to pathogenesis. Although amide-detected NMR experiments are widely employed in protein studies, 3JHNHα couplings between amide (1HN) and alpha (1Hα) protons impose an intrinsic limit on the achievable 1HN linewidth. Here, we present a homonuclear decoupling method that narrows the α-synuclein 1HN linewidths to 3-5 Hz. Tightly distributed 1JCαHα coupling values were employed to generate homogeneous antiphase coherences of 2HαzHNy and 4Hα(2)zHα(3)zHNy for nonglycine and glycine residues, respectively, which were combined with their in-phase HNy counterparts to achieve homonuclear decoupling. By reducing the multiplet structure to a singlet, the width of the 1HN cross-peak was reduced by ∼3-fold in the 2D HSQC and 3D intra-HNCA spectra, and good spectral quality was achieved without the need for postprocessing.


Assuntos
Ressonância Magnética Nuclear Biomolecular , alfa-Sinucleína/química , Amidas/química , Glicina/química , Prótons , Teoria Quântica , Soluções/química
8.
Nat Commun ; 10(1): 3070, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296852

RESUMO

CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/ultraestrutura , Guanilato Ciclase/ultraestrutura , Domínios Proteicos , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Microscopia Crioeletrônica , Guanilato Ciclase/genética , Guanilato Ciclase/imunologia , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Multimerização Proteica/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Transdução de Sinais/imunologia
9.
J Chem Phys ; 151(3): 034102, 2019 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-31325945

RESUMO

Nuclear magnetic resonance (NMR) is sensitive to dynamics on a wide range of correlation times. Recently, we have shown that analysis of relaxation rates via fitting to a correlation function with a small number of exponential terms could yield a biased characterization of molecular motion in solid-state NMR due to limited sensitivity of experimental data to certain ranges of correlation times. We introduced an alternative approach based on "detectors" in solid-state NMR, for which detector responses characterize motion for a range of correlation times and reduce potential bias resulting from the use of simple models for the motional correlation functions. Here, we show that similar bias can occur in the analysis of solution-state NMR relaxation data. We have thus adapted the detector approach to solution-state NMR, specifically separating overall tumbling motion from internal motions and accounting for contributions of chemical exchange to transverse relaxation. We demonstrate that internal protein motions can be described with detectors when the overall motion and the internal motions are statistically independent. We illustrate the detector analysis on ubiquitin with typical relaxation data sets recorded at a single high magnetic field or at multiple high magnetic fields and compare with results of model-free analysis. We also compare our methodology to LeMaster's method of dynamics analysis.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Termodinâmica
10.
Chem Commun (Camb) ; 55(66): 9761-9764, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31355386

RESUMO

The effect of ions on the structure and dynamics of a spider silk protein is elucidated. Chaotropic ions prevent intra- and inter-molecular interactions on the repetitive domain, which are required to maintain the solubility, while kosmotropic ions promote hydrogen bond interactions in the glycine-rich region, which are a prerequisite for ß-sheet formation.


Assuntos
Conformação Proteica em Folha beta , Seda/química , Animais , Cloretos/química , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/química , Sódio/química , Solubilidade , Aranhas
11.
Carbohydr Polym ; 220: 176-184, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31196538

RESUMO

A new glycosaminoglycan (LF-GAG) was purified from the slug Limacus flavus. Its unique chemical structure and heparanase inhibitory activity were studied in this work. The native LF-GAG was composed of L-iduronic acid (L-IdoA) and N-acetyl-D-glucosamine (D-GlcNAc), with a Mw of 22,700 Da. To elucidate the precise structure and structure-activity relationship, its deacetylation-deaminative depolymerized product (dLF-GAG) was prepared, and from which four oligosaccharides were purified. Combining the NMR spectral analysis of LF-GAG and its derived oligosaccharides, the structure of LF-GAG was deduced to be -4)-L-IdoA2R-(α1,4)-D-GlcNAc-(α1-, in which R was -OH (˜80%) or -OSO3- (˜20%). Bioactivity assays showed that LF-GAG could potently inhibit human heparanase (IC50, 0.10 µM). dLF-GAG and LF-3 were less potent but also active for heparanase inhibition. Structure-activity relationship analysis indicated that the chain length and sulfate substitution of LF-GAG are essential for its heparanase inhibitory activity.


Assuntos
Acetilglucosamina/química , Gastrópodes/metabolismo , Glucuronidase/antagonistas & inibidores , Glicosaminoglicanos , Ácido Idurônico/química , Animais , Glicosaminoglicanos/química , Glicosaminoglicanos/isolamento & purificação , Glicosaminoglicanos/farmacologia , Ressonância Magnética Nuclear Biomolecular/métodos
12.
Chem Commun (Camb) ; 55(55): 7899-7902, 2019 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-31199417

RESUMO

Fast-magic-angle-spinning solid-state NMR is a developing technique for determination of protein structure and dynamics. Proton-proton correlations usually lead to rough distance restraints, a serious hurdle towards high-resolution structures. Analogous to the "eNOE" concept in solution, an integrative approach for more accurate restraints enables improved structural accuracy with minimal analytical effort.


Assuntos
Anidrase Carbônica II/química , Espectrina/química , Animais , Galinhas , Humanos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Domínios de Homologia de src
13.
Nat Commun ; 10(1): 2574, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189925

RESUMO

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Domínios Proteicos/genética , Adenosina Trifosfatases/genética , Ácido Glutâmico/genética , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Relação Estrutura-Atividade , Tirosina/genética , Tirosina/metabolismo
14.
Nat Commun ; 10(1): 2511, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175284

RESUMO

Chemical shifts (CS) are determined from NMR experiments and represent the resonance frequency of the spin of atoms in a magnetic field. They contain a mixture of information, encompassing the in-solution conformations a protein adopts, as well as the movements it performs. Due to their intrinsically multi-faceted nature, CS are difficult to interpret and visualize. Classical approaches for the analysis of CS aim to extract specific protein-related properties, thus discarding a large amount of information that cannot be directly linked to structural features of the protein. Here we propose an autoencoder-based method, called ShiftCrypt, that provides a way to analyze, compare and interpret CS in their native, multidimensional space. We show that ShiftCrypt conserves information about the most common structural features. In addition, it can be used to identify hidden similarities between diverse proteins and peptides, and differences between the same protein in two different binding states.


Assuntos
Redes Neurais (Computação) , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/ultraestrutura , Aminoácidos , Fenômenos Biofísicos , Imagem por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Secundária de Proteína
15.
Phytochemistry ; 164: 192-205, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31174083

RESUMO

The chemical composition of five marine microalgae (Dunaliella sp., Dunaliella salina, Chaetoceros calcitrans, Chaetoceros gracilis and Tisochrysis lutea) was investigated through nuclear magnetic resonance (NMR) spectroscopic study of the soluble material obtained by sequential extraction with hexane, ethyl acetate (AcOEt) and methanol of biomass from stationary phase cultures. Hexane extracted the major lipids present in the microalgae during the stationary phase of growth, which correspond to storage lipids. Triacylglycerols (TGs) were the only storage lipids produced by Dunaliella and Chaetoceros. In contrast, T. lutea predominantly stored polyunsaturated long-chain alkenones, with sterols also detected as minor components of the hexane extract. The molecular structure of brassicasterol was determined in T. lutea and the presence of squalene in this sample was also unequivocally detected. Monogalactosyldiacylglycerols (MGDGs) and pigments were concentrated in the AcOEt extracts. C. calcitrans and D. salina constituted an exception due to the high amount of TGs and glycerol produced, respectively, by these two strains. Chlorophylls a and b and ß-carotene were the major pigments synthesized by Dunaliella and chlorophyll a and fucoxanthin were the only pigments detected in Chaetoceros and T. lutea. Information concerning the acyl chains present in TGs and MGDGs as well as the positional distribution of acyl chains on the glycerol moiety was obtained by NMR analysis of hexane and AcOEt extracts, with results consistent with those expected for the genera studied. Fatty acid composition of TGs in the two Dunaliella strains was different, with polyunsaturated acyl chains almost absent in the storage lipids produced by D. salina. Except in C. calcitrans, the polar nature of soluble compounds was inferred through the relative extraction yield using methanol as the extraction solvent. Glycerol was the major component of this fraction for the Dunaliella strains. In T. lutea 1,4/2,5-cyclohexanetetrol (CHT) and dimethylsulfoniopropionate (DMSP) preponderated. CHT was also the major polyol present in the Chaetoceros strains in which DMSP was not detected, but prominent signals of 2,3-dihydroxypropane-1-sulfonate (DHSP) were observed in the 1H NMR spectra of methanolic extracts. The presence of DHSP confirms the production of this metabolite by diatoms. In addition, several other minor compounds (digalactosyldiacyglycerols (DGDGs), sulphoquinovosyldiacylglycerols (SQDGs), amino acids, carbohydrates, scyllo-inositol, mannitol, lactic acid and homarine) were also identified in the methanolic extracts. The antibacterial and antibiofilm activities of the extracts were tested. The AcOEt extract from C. gracilis showed a moderate antibiofilm activity.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Microalgas/química , Ressonância Magnética Nuclear Biomolecular , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antifúngicos/química , Antifúngicos/isolamento & purificação , Fungos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Microalgas/metabolismo , Testes de Sensibilidade Microbiana , Conformação Molecular
16.
Chemphyschem ; 20(13): 1680-1689, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31087613

RESUMO

We employed deuterium solid-state NMR techniques under static conditions to discern the details of the µs-ms timescale motions in the flexible N-terminal subdomain of Aß1-40 amyloid fibrils, which spans residues 1-16. In particular, we utilized a rotating frame (R1ρ ) and the newly developed time domain quadrupolar Carr-Purcell-Meiboom-Gill (QCPMG) relaxation measurements at the selectively deuterated side chains of A2, H6, and G9. The two experiments are complementary in terms of probing somewhat different timescales of motions, governed by the tensor parameters and the sampling window of the magnetization decay curves. The results indicated two mobile "free" states of the N-terminal domain undergoing global diffusive motions, with isotropic diffusion coefficients of 0.7-1 ⋅ 108 and 0.3-3 ⋅ 106 ad2 s-1 . The free states are also involved in the conformational exchange with a single bound state, in which the diffusive motions are quenched, likely due to transient interactions with the structured hydrophobic core. The conformational exchange rate constants are 2-3 ⋅ 105  s-1 and 2-3 ⋅ 104  s-1 for the fast and slow diffusion free states, respectively.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Fragmentos de Peptídeos/química , Deutério , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos
17.
Chem Commun (Camb) ; 55(41): 5777-5780, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31041432

RESUMO

Investigating the interplay in a minimal redox complex of cytochrome-P450 and its reductase is crucial for understanding cytochrome-P450's enzymatic activity. Probing the hotspots of dynamic structural interactions using NMR revealed the engagement of loop residues from P450-reductase to be responsible for the enhanced affinity of CYP450 towards its obligate redox partner.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Mapas de Interação de Proteínas , Animais , Sistema Enzimático do Citocromo P-450/química , Humanos , Modelos Moleculares , NADPH-Ferri-Hemoproteína Redutase/química , Ressonância Magnética Nuclear Biomolecular/métodos , Oxirredução , Mapeamento de Interação de Proteínas/métodos , Coelhos
18.
J Chem Theory Comput ; 15(6): 3844-3853, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31042036

RESUMO

Configurational entropy change is a central constituent of the free energy change in noncovalent interactions between biomolecules. Due to both experimental and computational limitations, however, the impact of individual contributions to configurational entropy change remains underexplored. Here, we develop a novel, fully analytical framework to dissect the configurational entropy change of binding into contributions coming from molecular internal and external degrees of freedom. Importantly, this framework accounts for all coupled and uncoupled contributions in the absence of an external field. We employ our parallel implementation of the maximum information spanning tree algorithm to provide a comprehensive numerical analysis of the importance of the individual contributions to configurational entropy change on an extensive set of molecular dynamics simulations of protein binding processes. Contrary to commonly accepted assumptions, we show that different coupling terms contribute significantly to the overall configurational entropy change. Finally, while the magnitude of individual terms may be largely unpredictable a priori, the total configurational entropy change can be well approximated by rescaling the sum of uncoupled contributions from internal degrees of freedom only, providing support for NMR-based approaches for configurational entropy change estimation.


Assuntos
Entropia , Proteínas/química , Algoritmos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica
19.
Nat Commun ; 10(1): 2222, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110237

RESUMO

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl ß-D-glucoside and methyl 6-thio-ß-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-ß-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.


Assuntos
Domínio Catalítico , Glucosidases/metabolismo , Modelos Moleculares , Proteínas de Plantas/metabolismo , Biocatálise , Cristalografia por Raios X , Ensaios Enzimáticos/métodos , Glucosidases/química , Glucosidases/isolamento & purificação , Glicosídeos/metabolismo , Hordeum/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Plântula/metabolismo , Especificidade por Substrato
20.
Nat Commun ; 10(1): 2314, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127101

RESUMO

Histone methyltransferase MLL4 is centrally involved in transcriptional regulation and is often mutated in human diseases, including cancer and developmental disorders. MLL4 contains a catalytic SET domain that mono-methylates histone H3K4 and seven PHD fingers of unclear function. Here, we identify the PHD6 finger of MLL4 (MLL4-PHD6) as a selective reader of the epigenetic modification H4K16ac. The solution NMR structure of MLL4-PHD6 in complex with a H4K16ac peptide along with binding and mutational analyses reveal unique mechanistic features underlying recognition of H4K16ac. Genomic studies show that one third of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions in vivo and that MLL4 occupancy in a set of genomic targets depends on the acetyltransferase activity of MOF, a H4K16ac-specific acetyltransferase. The recognition of H4K16ac is conserved in the PHD7 finger of paralogous MLL3. Together, our findings reveal a previously uncharacterized acetyllysine reader and suggest that selective targeting of H4K16ac by MLL4 provides a direct functional link between MLL4, MOF and H4K16 acetylation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Dedos de Zinco PHD/fisiologia , Acetilação , Animais , Sítios de Ligação , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Técnicas de Inativação de Genes , Células HEK293 , Histona Acetiltransferases/genética , Histona-Lisina N-Metiltransferase/química , Histonas/química , Humanos , Camundongos Transgênicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
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