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1.
Nat Commun ; 12(1): 756, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531463

RESUMO

[FeFe]-hydrogenases are efficient H2-catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic Hox-state and the inactive but oxygen-resistant Hinact-state in [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii. The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O2-binding and consequently, cofactor degradation. This protection mechanism depends on three non-conserved amino acids situated approximately 13 Å away from the H-cluster, demonstrating that the 1st coordination sphere chemistry of the H-cluster can be remote-controlled by distant residues.


Assuntos
Cristalografia por Raios X/métodos , Sítios de Ligação , Domínio Catalítico , Clostridium beijerinckii/enzimologia , Clostridium beijerinckii/patogenicidade , Eletroquímica , Cinética , Modelos Teóricos , Espectroscopia de Infravermelho com Transformada de Fourier
2.
J Vis Exp ; (168)2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33616091

RESUMO

Here, a protocol is presented to facilitate the creation of large volumes (> 100 µL) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.


Assuntos
Ascomicetos/enzimologia , Ácido Aspártico Endopeptidases/química , Cristalografia por Raios X/métodos , Cristalografia por Raios X/normas , Proteínas Fúngicas/química , Cristalização
3.
Methods Mol Biol ; 2225: 125-162, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33108661

RESUMO

The myxoma virus has become of interest in human medicine in the last two decades as it has the ability to infect many types of human cancer cells and is being used as a platform to develop viro-therapeutic agents that suppress aggressive and damaging immune responses and inflammation. Furthermore, the myxoma virus encodes proteins that have strong immunosuppressive effects, and several of the myxoma virus-encoded immunomodulators are being developed to treat systemic inflammatory syndromes such as cardiovascular disease and transplant rejection. Myxoma virus encodes the M-T7 protein, the most abundantly secreted protein expressed in myxoma virus-infected cells, originally identified as a rabbit species-specific interferon-gamma (IFN-γ) receptor homolog and as a chemokine-modulating protein binding a wide range of mammalian chemokines. M-T7 is a critical virulence factor for viral pathogenesis that increases virus lethality when expressed. Although M-T7 has been extensively studied using biochemical and biophysical techniques and its interactome map is well known, its three-dimensional (3D) structure remains elusive. Obtaining the 3D structure of M-T7 would be greatly beneficial and is a crucial step toward advancing M-T7 research through understanding the molecular function and activity of M-T7 as a novel therapeutic reagent and to rationally develop this protein as a drug. This chapter provides an overview of the structural determination techniques, especially X-ray crystallography, that can be applied toward the goal of achieving the first high-resolution structure of M-T7. In addition, details of up-and-coming methods are discussed, including X-ray diffraction at X-ray free electron lasers (XFELs), nuclear magnetic resonance (NMR), cryo-electron microscopy (cryo-EM), Micro-electron diffraction (Micro-ED), and small-angle X-ray scattering (SAXS), and their potential applications to M-T7 structural biology.


Assuntos
Cristalização/métodos , Cristalografia por Raios X/métodos , Myxoma virus/química , Receptores de Interferon/ultraestrutura , Proteínas Virais/ultraestrutura , Fatores de Virulência/genética , Difração de Raios X/métodos , Sequência de Aminoácidos , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X/instrumentação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Receptores de Interferon/química , Receptores de Interferon/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestrutura , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/genética , Fatores de Virulência/metabolismo , Difração de Raios X/instrumentação
4.
Nat Commun ; 11(1): 5695, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33173051

RESUMO

Cytoplasmic dynein-1 (dynein) is the motor responsible for most retrograde transport of cargoes along microtubules in eukaryotic cells, including organelles, mRNA and viruses. Cargo selectivity and activation of processive motility depend on a group of so-called "activating adaptors" that link dynein to its general cofactor, dynactin, and cargoes. The mechanism by which these adaptors regulate dynein transport is poorly understood. Here, based on crystal structures, quantitative binding studies, and in vitro motility assays, we show that BICD2, CRACR2a, and HOOK3, representing three subfamilies of unrelated adaptors, interact with the same amphipathic helix of the dynein light intermediate chain-1 (LIC1). While the hydrophobic character of the interaction is conserved, the three adaptor subfamilies use different folds (coiled-coil, EF-hand, HOOK domain) and different surface contacts to bind the LIC1 helix with affinities ranging from 1.5 to 15.0 µM. We propose that a tunable LIC1-adaptor interaction modulates dynein's motility in a cargo-specific manner.


Assuntos
Transporte Biológico/fisiologia , Dineínas do Citoplasma/metabolismo , Proteínas Motores Moleculares/metabolismo , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular , Cristalografia por Raios X/métodos , Complexo Dinactina/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica
5.
J Vis Exp ; (163)2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-33044452

RESUMO

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to >10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.


Assuntos
Cristalografia por Raios X/métodos , Síncrotrons , Viscosidade/efeitos dos fármacos , Austrália , Injeções
6.
PLoS One ; 15(9): e0239706, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32976530

RESUMO

Flash-cooling of macromolecular crystals for X-ray diffraction analysis is usually performed in liquid nitrogen (LN2). Cryogens different than LN2 are used as well for this procedure but are highly underrepresented, e.g., liquid propane and liquid ethane. These two cryogens have significantly higher cooling rates compared with LN2 and may thus be beneficial for flash-cooling of macromolecular crystals. Flash-cooling in liquid propane or liquid ethane results in sample vitrification but is accompanied by solidification of these cryogens, which is not compatible with the robotic systems nowadays used for crystal mounting at most synchrotrons. Here we provide a detailed description of a new double-chambered device and procedure to flash-cool loop mounted macromolecular crystals in different cryogenic liquids. The usage of this device may result in specimens of better crystal- and optical quality in terms of mosaic spread and ice contamination. Furthermore, applying the described procedure with the new double-chambered device provides the possibility to screen for the best flash-cooling cryogen for macromolecular crystals on a routine basis, and, most importantly, the samples obtained allow the usage of state-of-the-art robotic sample-loading systems at synchrotrons.


Assuntos
Temperatura Baixa , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Etano/química , Nitrogênio/química , Propano/química
7.
Proc Natl Acad Sci U S A ; 117(37): 22944-22952, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868441

RESUMO

γδ T cells form an abundant part of the human cellular immune system, where they respond to tissue damage, infection, and cancer. The spectrum of known molecular targets recognized by Vδ1-expressing γδ T cells is becoming increasingly diverse. Here we describe human γδ T cells that recognize CD1b, a lipid antigen-presenting molecule, which is inducibly expressed on monocytes and dendritic cells. Using CD1b tetramers to study multiple donors, we found that many CD1b-specific γδ T cells use Vδ1. Despite their common use of Vδ1, three CD1b-specific γδ T cell receptors (TCRs) showed clear differences in the surface of CD1b recognized, the requirement for lipid antigens, and corecognition of butryophilin-like proteins. Several Vγ segments were present among the CD1b-specific TCRs, but chain swap experiments demonstrated that CD1b specificity was mediated by the Vδ1 chain. One of the CD1b-specific Vδ1+ TCRs paired with Vγ4 and shows dual reactivity to CD1b and butyrophilin-like proteins. αß TCRs typically recognize the peptide display platform of MHC proteins. In contrast, our results demonstrate the use of rearranged receptors to mediate diverse modes of recognition across the surface of CD1b in ways that do and do not require carried lipids.


Assuntos
Antígenos CD1/metabolismo , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismo , Apresentação do Antígeno , Antígenos CD1/imunologia , Cristalografia por Raios X/métodos , Humanos , Linfócitos Intraepiteliais/fisiologia , Lipídeos/imunologia , Ativação Linfocitária/imunologia , Modelos Moleculares , Monócitos/metabolismo , Linfócitos T/imunologia
8.
PLoS One ; 15(8): e0238150, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866159

RESUMO

Immunogenicity is an important concern for therapeutic antibodies during drug development. By analyzing co-crystal structures of idiotypic antibodies and their antibodies, we found that anti-idiotypic antibodies usually bind the Complementarity Determining Regions (CDR) of idiotypic antibodies. Sequence and structural features were identified for distinguishing immunogenic antibodies from non-immunogenic antibodies. For example, non-immunogenic antibodies have a significantly larger cavity volume at the CDR region and a more hydrophobic CDR-H3 loop than immunogenic antibodies. Antibodies containing no Gly at the turn of CDR-H2 loop are often immunogenic. We integrated these features together with a machine learning platform to Predict Immunogenicity for humanized and full human THerapeutic Antibodies (PITHA). This method achieved an accuracy of 83% in leave-one-out experiment for 29 therapeutic antibodies with available crystal structures. The accuracy decreased to 65% for 23 test antibodies with modeled structures, because their crystal structures were not available, and the prediction was made with modeled structures. The server of this method is accessible at http://mabmedicine.com/PITHA.


Assuntos
Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Formação de Anticorpos/imunologia , Cristalografia por Raios X/métodos , Desenvolvimento de Medicamentos/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica
9.
J Med Chem ; 63(15): 8043-8045, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32786236

RESUMO

Inhibitors of the respiratory syncytial virus (RSV) fusion protein block entry of the virus into the cell and have shown varying efficacy in a human challenge model of RSV disease. Trials in patient populations are yet to show significant benefits. Jonckers et al. ( J. Med. Chem. 2020, DOI: 10.1021/acs.jmedchem.0c00226) describe the discovery of JNJ-53718678 which can now claim the leading position in clinical evaluation. For RSV inhibitors, the current status of the clinical development of the compound is discussed.


Assuntos
Antivirais/química , Imidazolidinas/química , Indóis/química , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Inibidores de Proteínas Virais de Fusão/química , Antivirais/farmacologia , Antivirais/uso terapêutico , Cristalografia por Raios X/métodos , Humanos , Imidazolidinas/farmacologia , Imidazolidinas/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/fisiologia , Resultado do Tratamento , Inibidores de Proteínas Virais de Fusão/farmacologia
10.
AAPS PharmSciTech ; 21(7): 241, 2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839899

RESUMO

Rutin has many biological activities, but poor solubility and absorption limit its oral application. This study aimed to investigate the characterization of metastable rutin-ethanolate and its solubility and oral bioavailability. In this research, a soluble rutin/CH3CH2OH solvate (Form Π) was prepared by solvent crystallization. High-performance liquid chromatography, gas chromatograph, and 1H-NMR showed that Form Π was formed by rutin and ethanol in a molar ratio of 1:1. The changes of Fourier transform infrared spectroscopy and 1H-NMR spectrum and the density functional theory (DFT) calculation predicted hydrogen bond formation between 4'-O of rutin and -OH of ethanol. The results of morphology, solid state CP/MAS 13C-NMR, X-ray diffraction, and differential scanning calorimetry (DSC) revealed that Form Π is a novel polymorph that differs from Form Ι (rutin trihydrate). Form Π can be stored for a long time under sealed and dry conditions at 40°C but would gradually transform into Form Ι under humid conditions. Although Form Π is a new metastable polymorph relative to Form Ι, Form Π has better solubility and faster dissolution rate. Moreover, the bioavailability of Form Π was 2.04 times higher than that of Form Ι. This outcome implied that Form Π would be a prospective raw material of oral preparation.


Assuntos
Rutina/química , Administração Oral , Disponibilidade Biológica , Varredura Diferencial de Calorimetria , Cristalografia por Raios X/métodos , Etanol/química , Espectroscopia de Ressonância Magnética , Estudos Prospectivos , Rutina/farmacocinética , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
11.
Nat Commun ; 11(1): 4248, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843623

RESUMO

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time. We find that the photocycle kinetics and structural dynamics of PYP in the crystalline form deviate from those in solution from the very first steps following photon absorption. This illustrates that ultrafast TRC results cannot be uncritically extrapolated to in vivo function, and that comparative spectroscopic experiments on proteins in crystalline and solution states can help identify structural intermediates under native conditions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X/métodos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Proteínas de Bactérias/efeitos da radiação , Cinética , Luz , Estrutura Molecular , Processos Fotoquímicos , Fotorreceptores Microbianos/efeitos da radiação , Conformação Proteica , Análise Espectral
12.
PLoS One ; 15(6): e0235341, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32603354

RESUMO

Hydroxynitrile lyases (HNL's) belonging to the α/ß-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/ß-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/ß-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.


Assuntos
Aldeído Liases/química , Aldeído Liases/genética , Domínio Catalítico , Cristalografia por Raios X/métodos , Esterases/química , Esterases/genética , Hevea/genética , Hevea/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida/métodos , Proteínas de Plantas/genética , Dobramento de Proteína , Especificidade por Substrato , Tabaco/genética , Tabaco/metabolismo
13.
Sci Rep ; 10(1): 10205, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576842

RESUMO

Serine-based ß-lactamases of Class A, C and D all rely on a key water molecule to hydrolyze and inactivate ß-lactam antibiotics. This process involves two conserved catalytic steps. In the first acylation step, the ß-lactam antibiotic forms an acyl-enzyme intermediate (ES*) with the catalytic serine residue. In the second deacylation step, an activated water molecule serves as nucleophile (WAT_Nu) to attack ES* and release the inactivated ß-lactam. The coordination and activation of WAT_Nu is not fully understood. Using time-resolved x-ray crystallography and QM/MM simulations, we analyzed three intermediate structures of Class A ß-lactamase PenP as it slowly hydrolyzed cephaloridine. WAT_Nu is centrally located in the apo structure but becomes slightly displaced away by ES* in the post-acylation structure. In the deacylation structure, WAT_Nu moves back and is positioned along the Bürgi-Dunitz trajectory with favorable energetic profile to attack ES*. Unexpectedly, WAT_Nu is also found to adopt a catalytically incompetent conformation in the deacylation structure forming a hydrogen bond with ES*. Our results reveal that ES* plays a significant role in coordinating and activating WAT_Nu through subtle yet distinct interactions at different stages of the catalytic process. These interactions may serve as potential targets to circumvent ß-lactamase-mediated antibiotic resistance.


Assuntos
Água/química , Água/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Acilação , Antibacterianos/química , Antibacterianos/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X/métodos , Ligação de Hidrogênio , Hidrólise , Cinética , Serina/química , Serina/metabolismo , beta-Lactamas/química , beta-Lactamas/metabolismo
14.
Nat Commun ; 11(1): 2319, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385234

RESUMO

Bruton's tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, Btk signaling sustains growth of several B-cell neoplasms which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the structural mechanism by which certain XLA mutations in the SH2 domain strongly perturb Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain required for Btk activation and to which multiple XLA mutations map. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevents activation of wild-type and TKI-resistant Btk, inhibiting Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Cristalografia por Raios X/métodos , Linfoma/metabolismo , Tirosina Quinase da Agamaglobulinemia/genética , Western Blotting , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Dicroísmo Circular , Citometria de Fluxo , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Linfoma/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação/genética
15.
J Med Chem ; 63(10): 5185-5200, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32364386

RESUMO

Human carbonic anhydrase (CA; EC 4.2.1.1) isoforms II and VII are implicated in neuronal excitation, seizures, and neuropathic pain (NP). Their selective inhibition over off-target CAs is expected to produce an anti-NP action devoid of side effects due to promiscuous CA modulation. Here, a drug design strategy based on the observation of (dis)similarities between the target CA active sites was planned with benzenesulfonamide derivatives and, for the first time, a phosphorus-based linker. Potent and selective CA II/VII inhibitors were identified among the synthesized phenyl(thio)phosphon(amid)ates 3-22. X-ray crystallography depicted the binding mode of phosphonic acid 3 to both CAs II and VII. The most promising derivatives, after evaluation of their stability in acidic media, were tested in a mouse model of oxaliplatin-induced neuropathy. The most potent compound racemic mixture was subjected to HPLC enantioseparation, and the identification of the eutomer, the (S)-enantiomer, allowed to halve the dose totally relieving allodynia in mice.


Assuntos
Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/uso terapêutico , Anidrases Carbônicas/metabolismo , Hiperalgesia/tratamento farmacológico , Oxaliplatina/toxicidade , Animais , Antineoplásicos/toxicidade , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Temperatura Baixa/efeitos adversos , Cristalografia por Raios X/métodos , Modelos Animais de Doenças , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/enzimologia , Masculino , Camundongos , Neuralgia/induzido quimicamente , Neuralgia/tratamento farmacológico , Neuralgia/enzimologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico
16.
Nat Methods ; 17(7): 681-684, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32451477

RESUMO

Time-resolved crystallography with X-ray free-electron lasers enables structural characterization of light-induced reactions on ultrafast timescales. To be biologically and chemically relevant, such studies must be carried out in an appropriate photoexcitation regime to avoid multiphoton artifacts, a common issue in recent studies. We describe numerical and experimental approaches to determine how many photons are needed for single-photon excitation in microcrystals, taking into account losses by scattering.


Assuntos
Cristalografia por Raios X/métodos , Fótons , Radiação Eletromagnética , Lasers , Luz , Espalhamento de Radiação
17.
Nat Chem Biol ; 16(8): 920-929, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32451508

RESUMO

The fundamental and assorted roles of ß-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on ß-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/ß)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical ß-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of ß-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of ß-1,3-glucans, which can be exploited for biotechnological applications.


Assuntos
Glucana 1,3-beta-Glucosidase/química , Glicosídeo Hidrolases/química , beta-Glucanas/química , Sequência de Aminoácidos/genética , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Glucana 1,3-beta-Glucosidase/metabolismo , Glucanos/química , Glicosídeos/química , Modelos Moleculares , Especificidade por Substrato/fisiologia
18.
Mol Cell ; 78(5): 903-914.e4, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32396821

RESUMO

LSD1 (lysine specific demethylase; also known as KDM1A), the first histone demethylase discovered, regulates cell-fate determination and is overexpressed in multiple cancers. LSD1 demethylates histone H3 Lys4, an epigenetic mark for active genes, but requires the CoREST repressor to act on nucleosome substrates. To understand how an accessory subunit (CoREST) enables a chromatin enzyme (LSD1) to function on a nucleosome and not just histones, we have determined the crystal structure of the LSD1/CoREST complex bound to a 191-bp nucleosome. We find that the LSD1 catalytic domain binds extranucleosomal DNA and is unexpectedly positioned 100 Å away from the nucleosome core. CoREST makes critical contacts with both histone and DNA components of the nucleosome, explaining its essential function in demethylating nucleosome substrates. Our studies also show that the LSD1(K661A) frequently used as a catalytically inactive mutant in vivo (based on in vitro peptide studies) actually retains substantial H3K4 demethylase activity on nucleosome substrates.


Assuntos
Histona Desmetilases/metabolismo , Histona Desmetilases/ultraestrutura , Sequência de Aminoácidos , Domínio Catalítico , Cromatina/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Cristalografia por Raios X/métodos , DNA/genética , DNA/metabolismo , Histona Desmetilases/genética , Histonas/metabolismo , Humanos , Metilação , Modelos Moleculares , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica
19.
J Med Chem ; 63(15): 8046-8058, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32407115

RESUMO

Respiratory syncytial virus (RSV) is a seasonal virus that infects the lungs and airways of 64 million children and adults every year. It is a major cause of acute lower respiratory tract infection and is associated with significant morbidity and mortality. Despite the large medical and economic burden, treatment options for RSV-associated bronchiolitis and pneumonia are limited and mainly consist of supportive care. This publication covers the medicinal chemistry efforts resulting in the identification of JNJ-53718678, an orally bioavailable RSV inhibitor that was shown to be efficacious in a phase 2a challenge study in healthy adult subjects and that is currently being evaluated in hospitalized infants and adults. Cocrystal structures of several new derivatives helped in rationalizing some of the structure-activity relationship (SAR) trends observed.


Assuntos
Antivirais/química , Descoberta de Drogas/métodos , Imidazolidinas/química , Indóis/química , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Inibidores de Proteínas Virais de Fusão/química , Administração Oral , Antivirais/administração & dosagem , Cristalografia por Raios X/métodos , Células HeLa , Humanos , Imidazolidinas/administração & dosagem , Indóis/administração & dosagem , Estrutura Secundária de Proteína , Vírus Sincicial Respiratório Humano/fisiologia , Inibidores de Proteínas Virais de Fusão/administração & dosagem
20.
RNA ; 26(8): 1049-1059, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32371454

RESUMO

Nop9 is an essential factor in the processing of preribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its target RNA within the 20S preribosomal RNA. This structure reveals that Nop9 brings together a carboxy-terminal module recognizing the 5' single-stranded region of the RNA and a bifunctional amino-terminal module recognizing the central double-stranded stem region. We further show that the 3' single-stranded region of the 20S target RNA adds sequence-independent binding energy to the RNA-Nop9 interaction. Both the amino- and carboxy-terminal modules retain the characteristic sequence-specific recognition of PUF proteins, but the amino-terminal module has also evolved a distinct interface, which allows Nop9 to recognize either single-stranded RNA sequences or RNAs with a combination of single-stranded and structured elements.


Assuntos
RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X/métodos , Humanos , Conformação Proteica em alfa-Hélice/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
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