RESUMO
Environmental allergens, including fungi, insects and mites, trigger type 2 immunity; however, the innate sensing mechanisms and initial signaling events remain unclear. Herein, we demonstrate that allergens trigger RIPK1-caspase 8 ripoptosome activation in epithelial cells. The active caspase 8 subsequently engages caspases 3 and 7, which directly mediate intracellular maturation and release of IL-33, a pro-atopy, innate immunity, alarmin cytokine. Mature IL-33 maintained functional interaction with the cognate ST2 receptor and elicited potent pro-atopy inflammatory activity in vitro and in vivo. Inhibiting caspase 8 pharmacologically and deleting murine Il33 and Casp8 each attenuated allergic inflammation in vivo. Clinical data substantiated ripoptosome activation and IL-33 maturation as likely contributors to human allergic inflammation. Our findings reveal an epithelial barrier, allergen-sensing mechanism that converges on the ripoptosome as an intracellular molecular signaling platform, triggering type 2 innate immune responses. These findings have significant implications for understanding and treating human allergic diseases.
Assuntos
Alérgenos/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Adolescente , Animais , Caspase 8/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Criança , Pré-Escolar , Citocinas/imunologia , Células Epiteliais/imunologia , Feminino , Células HEK293 , Humanos , Hipersensibilidade/imunologia , Interleucina-33/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologiaRESUMO
The gene product of the open reading frame Rv3340 from Mycobacterium tuberculosis is annotated as encoding a probable O-acetylhomoserine (OAH) sulfhydrylase (MetC), an enzyme that catalyzes the last step in the biosynthesis of methionine, which is an essential amino acid in bacteria and plants. Following overexpression in Escherichia coli, the M. tuberculosis MetC enzyme was purified and crystallized using the hanging-drop vapor-diffusion method. Native diffraction data were collected from crystals belonging to space group P2(1) and were processed to a resolution of 2.1â Å.
Assuntos
Carbono-Oxigênio Liases/química , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/isolamento & purificação , Sequência Conservada , Cristalografia por Raios X , Expressão Gênica , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Biotinylation identification (BioID) is a method designed to provide new cellular location and functional knowledge of the protein of interest through the identification of those proteins surrounding and in direct contact. A biotin ligase is fused onto the protein of interest and expressed in cells where it can biotinylate even short-lived transient protein complexes. In addition, due to the proximity labeling nature of the experiment, cellular localization and functional enrichment information can also be obtained. Since labeling occurs only after the addition of biotin, temporal relationships and localization changes (e.g., cytoplasmic to nuclear) can also be identified. Labeled proteins are easily purified, and contaminants minimized, using the strong interaction between biotin and streptavidin. Mass spectrometry analysis of the purified proteins allows for the identification of potential interactors for further validation and characterization.
Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Mapas de Interação de Proteínas , Proteômica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Métodos Analíticos de Preparação de Amostras , Animais , Biotinilação , Carbono-Nitrogênio Ligases/genética , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genética , Fatores de TempoRESUMO
Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease with a complex underlying genetic etiology. Herein, we conduct whole-exome sequencing of a multigeneration EoE pedigree (discovery set) and 61 additional multiplex families with EoE (replication set). A series of rare, heterozygous, missense variants are identified in the genes encoding the desmosome-associated proteins DSP and PPL in 21% of the multiplex families. Esophageal biopsies from patients with these variants retain dilated intercellular spaces and decrease DSP and PPL expression even during disease remission. These variants affect barrier integrity, cell motility and RhoGTPase activity in esophageal epithelial cells and have increased susceptibility to calpain-14-mediated degradation. An acquired loss of esophageal DSP and PPL is present in non-familial EoE. Taken together, herein, we uncover a pathogenic role for desmosomal dysfunction in EoE, providing a deeper mechanistic understanding of tissue-specific allergic responses.
Assuntos
Desmoplaquinas/genética , Esofagite Eosinofílica/genética , Mucosa Esofágica/patologia , Plaquinas/genética , Adolescente , Biópsia , Calpaína/metabolismo , Estudos de Casos e Controles , Criança , Análise Mutacional de DNA , Desmoplaquinas/metabolismo , Desmossomos/patologia , Esofagite Eosinofílica/patologia , Mucosa Esofágica/citologia , Feminino , Células HEK293 , Células HaCaT , Heterozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Plaquinas/metabolismo , Proteólise , RNA-Seq , Análise de Célula Única , Sequenciamento do ExomaRESUMO
The crystal structure of the urease gamma subunit (UreA) from Mycobacterium tuberculosis, Rv1848, has been determined at 1.8 A resolution. The asymmetric unit contains three copies of Rv1848 arranged into a homotrimer that is similar to the UreA trimer in the structure of urease from Klebsiella aerogenes. Small-angle X-ray scattering experiments indicate that the Rv1848 protein also forms trimers in solution. The observed homotrimer and the organization of urease genes within the M. tuberculosis genome suggest that M. tuberculosis urease has the (alphabetagamma)(3) composition observed for other bacterial ureases. The gamma subunit may be of primary importance for the formation of the urease quaternary structure.
Assuntos
Mycobacterium tuberculosis/enzimologia , Urease/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Eosinophilic esophagitis (EoE) is a chronic, food antigen-driven, inflammatory disease of the esophagus and is associated with impaired barrier function. Evidence is emerging that loss of esophageal expression of the serine peptidase inhibitor, kazal type 7 (SPINK7), is an upstream event in EoE pathogenesis. Here, we provide evidence that loss of SPINK7 mediates its pro-EoE effects via kallikrein 5 (KLK5) and its substrate, protease-activated receptor 2 (PAR2). Overexpression of KLK5 in differentiated esophageal epithelial cells recapitulated the effect of SPINK7 gene silencing, including barrier impairment and loss of desmoglein-1 expression. Conversely, KLK5 deficiency attenuated allergen-induced esophageal protease activity, modified commensal microbiome composition, and attenuated eosinophilia in a murine model of EoE. Inhibition of PAR2 blunted the cytokine production associated with loss of SPINK7 in epithelial cells and attenuated the allergen-induced esophageal eosinophilia in vivo. Clinical samples substantiated dysregulated PAR2 expression in the esophagus of patients with EoE, and delivery of the clinically approved drug α1 antitrypsin (A1AT, a protease inhibitor) inhibited experimental EoE. These findings demonstrate a role for the balance between KLK5 and protease inhibitors in the esophagus and highlight EoE as a protease-mediated disease. We suggest that antagonizing KLK5 and/or PAR2 has potential to be therapeutic for EoE.
Assuntos
Esofagite Eosinofílica , Animais , Esofagite Eosinofílica/tratamento farmacológico , Células Epiteliais , Humanos , Calicreínas , Camundongos , Receptor PAR-2RESUMO
A novel series of c-jun N-terminal kinase (JNK) inhibitors were designed and developed from a high-throughput-screening hit. Through the optimization of the piperazine amide 1, several potent compounds were discovered. The X-ray crystal structure of 4g showed a unique binding mode different from other well known JNK3 inhibitors.
Assuntos
Amidas/síntese química , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Piperazinas/síntese química , Amidas/farmacologia , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Piperazinas/farmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-AtividadeRESUMO
Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 +/- 27 nM and 336 +/- 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 +/- 7.8 nM and 150 +/- 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z' values during the HTS campaign (0.84 +/- 0.03; 0.72 +/- 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Medições Luminescentes/métodos , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Sequência de Bases , Primers do DNA/genética , Humanos , Técnicas In Vitro , Luciferases , Miniaturização , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Quinases Associadas a rho/genéticaRESUMO
IL-33 is an epithelium-derived, pro-inflammatory alarmin with enigmatic nuclear localization and chromatin binding. Here we report the functional properties of nuclear IL-33. Overexpression of IL-33 does not alter global gene expression in transduced epithelial cells. Fluorescence recovery after photobleaching data show that the intranuclear mobility of IL-33 is ~10-fold slower than IL-1α, whereas truncated IL-33 lacking chromatin-binding activity is more mobile. WT IL-33 is more resistant to necrosis-induced release than truncated IL-33 and has a relatively slow, linear release over time after membrane dissolution as compared to truncated IL-33 or IL-1α. Lastly, IL-33 and histones are released as a high-molecular weight complex and synergistically activate receptor-mediated signaling. We thus propose that chromatin binding is a post-translational mechanism that regulates the releasability and ST2-mediated bioactivity of IL-33 and provide a paradigm to further understand the enigmatic functions of nuclear cytokines.
Assuntos
Cromatina/metabolismo , Citocinas/metabolismo , Espaço Extracelular/metabolismo , Interleucina-33/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Cinética , Modelos Biológicos , Necrose , Ligação ProteicaRESUMO
A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9 A resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54 A. N-terminal amino-acid sequencing indicates that the crystals contain a C-terminal fragment of the protein.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Corrinoides/química , Bactérias Gram-Positivas/enzimologia , Metanol/farmacologia , Acetilcoenzima A/biossíntese , Acetilcoenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Bactérias Gram-Positivas/metabolismo , Metanol/metabolismoRESUMO
VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 Å resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA. The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg²âº ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg²âº ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 Å closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Magnésio/química , Glicoproteínas de Membrana/química , Mycobacterium tuberculosis/metabolismo , RNA/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Domínio Catalítico , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Bacterianos , Magnésio/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Estrutura Terciária de Proteína , Subunidades Proteicas , RNA/química , Alinhamento de SequênciaRESUMO
The TB Structural Genomics Consortium is a worldwide organization of collaborators whose mission is the comprehensive structural determination and analyses of Mycobacterium tuberculosis proteins to ultimately aid in tuberculosis diagnosis and treatment. Congruent to the overall vision, Consortium members have additionally established an integrated facilities core to streamline M. tuberculosis structural biology and developed bioinformatics resources for data mining. This review aims to share the latest Consortium developments with the TB community, including recent structures of proteins that play significant roles within M. tuberculosis. Atomic resolution details may unravel mechanistic insights and reveal unique and novel protein features, as well as important protein-protein and protein-ligand interactions, which ultimately lead to a better understanding of M. tuberculosis biology and may be exploited for rational, structure-based therapeutics design.
Assuntos
Genômica/métodos , Cooperação Internacional , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/química , Cristalografia por Raios X , Bases de Dados de Proteínas , Desenho de Fármacos , Genoma Bacteriano , Genômica/tendências , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismoRESUMO
Given the significant body of data supporting an essential role for c-jun-N-terminal kinase (JNK) in neurodegenerative disorders, we set out to develop highly selective JNK inhibitors with good cell potency and good brain penetration properties. The structure-activity relationships (SAR) around a series of aminopyrimidines were evaluated utilizing biochemical and cell-based assays to measure JNK inhibition and brain penetration in mice. Microsomal stability in three species, P450 inhibition, inhibition of generation of reactive oxygen species (ROS), and pharmacokinetics in rats were also measured. Compounds 9g, 9i, 9j, and 9l had greater than 135-fold selectivity over p38, and cell-based IC(50) values < 100 nM. Moreover, compound 9l showed an IC(50) = 0.8 nM for inhibition of ROS and had good pharmacokinetic properties in rats along with a brain-to-plasma ratio of 0.75. These results suggest that biaryl substituted aminopyrimidines represented by compound 9l may serve as the first small molecule inhibitors to test efficacy of JNK inhibitors in neurodegenerative disorders.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Animais , Cristalografia por Raios X , Desenho de Fármacos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-AtividadeRESUMO
c-Jun N-terminal kinase 3alpha1 (JNK3alpha1) is a mitogen-activated protein kinase family member expressed primarily in the brain that phosphorylates protein transcription factors, including c-Jun and activating transcription factor-2 (ATF-2) upon activation by a variety of stress-based stimuli. In this study, we set out to design JNK3-selective inhibitors that had >1000-fold selectivity over p38, another closely related mitogen-activated protein kinase family member. To do this we employed traditional medicinal chemistry principles coupled with structure-based drug design. Inhibitors from the aminopyrazole class, such as SR-3576, were found to be very potent JNK3 inhibitors (IC(50) = 7 nm) with >2800-fold selectivity over p38 (p38 IC(50) > 20 microm) and had cell-based potency of approximately 1 microm. In contrast, indazole-based inhibitors exemplified by SR-3737 were potent inhibitors of both JNK3 (IC(50) = 12 nm) and p38 (IC(50) = 3 nm). These selectivity differences between the indazole class and the aminopyrazole class came despite nearly identical binding (root mean square deviation = 0.33 A) of these two compound classes to JNK3. The structural features within the compounds giving rise to the selectivity in the aminopyrazole class include the highly planar nature of the pyrazole, N-linked phenyl structures, which better occupied the smaller active site of JNK3 compared with the larger active site of p38.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteína Quinase 10 Ativada por Mitógeno/antagonistas & inibidores , Pirazóis/química , Pirazóis/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Fator 2 Ativador da Transcrição/metabolismo , Animais , Células Cultivadas , Cristalografia por Raios X , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Conformação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
Replication protein A (RPA) is the ubiquitous, eukaryotic single-stranded DNA (ssDNA) binding protein and is essential for DNA replication, recombination, and repair. Here, crystal structures of the soluble RPA heterodimer, composed of the RPA14 and RPA32 subunits, have been determined for the full-length protein in multiple crystal forms. In all crystals, the electron density for the N-terminal (residues 1-42) and C-terminal (residues 175-270) regions of RPA32 is weak and of poor quality indicating that these regions are disordered and/or assume multiple positions in the crystals. Hence, the RPA32 N terminus, that is hyperphosphorylated in a cell-cycle-dependent manner and in response to DNA damaging agents, appears to be inherently disordered in the unphosphorylated state. The C-terminal, winged helix-loop-helix, protein-protein interaction domain adopts several conformations perhaps to facilitate its interaction with various proteins. Although the ordered regions of RPA14/32 resemble the previously solved protease-resistant core crystal structure, the quaternary structures between the heterodimers are quite different. Thus, the four-helix bundle quaternary assembly noted in the original core structure is unlikely to be related to the quaternary structure of the intact heterotrimer. An organic ligand binding site between subunits RPA14 and RPA32 was identified to bind dioxane. Comparison of the ssDNA binding surfaces of RPA70 with RPA14/32 showed that the lower affinity of RPA14/32 can be attributed to a shallower binding crevice with reduced positive electrostatic charge.
Assuntos
DNA de Cadeia Simples/química , Modelos Moleculares , Subunidades Proteicas/química , Proteína de Replicação A/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dioxanos/química , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
The structure-based design and synthesis of a novel series of c-Jun N-terminal kinase (JNK) inhibitors with selectivity against p38 is reported. The unique structure of 3,5-disubstituted quinolines (2) was developed from the previously reported 4-(2,7-phenanthrolin-9-yl)phenol (1). The X-ray crystal structure of 16a in JNK3 reveals an unexpected binding mode for this new scaffold with protein.
Assuntos
Desenho de Fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Quinolinas/química , Quinolinas/farmacologia , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Estrutura Molecular , Fenantrolinas/síntese química , Fenantrolinas/química , Fenantrolinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The open-reading frame PF0895 in the genome of the hyperthermophilic archaeon, Pyrococcus furiosus, encodes a 206-residue protein (M(R )23,152). The structure of the recombinant protein was solved by single isomorphous replacement with anomalous scattering (SIRAS) using a mercury derivative. It has been refined to 1.70 A with a crystallographic R and R(free )values of 19.7% and 22.3%, respectively. The PF0895 structure is similar to those of the ATP binding cassettes observed in the ABC transporter family. However, bioinformatics and molecular analyses indicate that PF0895 is not part of the expected five-gene operon that encodes a typical prokaryotic solute-binding ABC transporter. Rather, transcriptional profiling data show that PF0895 is part of a novel four-gene operon (PF0895-PF0896-PF0897-PF0897.1) where only PF0895 has homologs in other organisms. Interestingly, from genome analysis, P. furiosus itself contains a second version of this complex, encoded by PF1090-PF1093. From the structural studies we can only conclude that one of the subunits of this novel membrane complex, PF0895, and its homolog PF1090, likely bind a purine nucleotide. PF0895 is therefore predicted to be part of a membrane-bound multiprotein complex unrelated to ABC transporters that is so far unique to P. furiosus. It appears to play a role in the stress response, as its expression is down regulated when the organism is subjected to cold-shock, where cells are transferred from 95 degrees C, near the optimal growth temperature, to 72 degrees C, near the minimal growth temperature. The related PF1090-containing operon is unaffected by cold-shock and is independently regulated.
Assuntos
Regulação da Expressão Gênica em Archaea , Complexos Multiproteicos/química , Proteínas de Transporte de Nucleotídeos/química , Proteínas/química , Pyrococcus furiosus/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Cristalografia por Raios X , Genoma Arqueal , Genômica , Modelos Biológicos , Conformação Molecular , Dados de Sequência Molecular , Proteínas de Transporte de Nucleotídeos/fisiologia , Conformação Proteica , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Using a high degree of automation, the crystallography core at the Southeast Collaboratory for Structural Genomics (SECSG) has developed a high-throughput protein-to-structure pipeline. Various robots and automation procedures have been adopted and integrated into a pipeline that is capable of screening 40 proteins for crystallization and solving four protein structures per week. This pipeline is composed of three major units: crystallization, structure determination/validation and crystallomics. Coupled with the protein-production cores at SECSG, the protein-to-structure pipeline provides a two-tiered approach for protein production at SECSG. In tier 1, all protein samples supplied by the protein-production cores pass through the pipeline using standard crystallization screening and optimization procedures. The protein targets that failed to yield diffraction-quality crystals (resolution better than 3.0 A) become tier 2 or salvaging targets. The goal of tier 2 target salvaging, carried out by the crystallomics core, is to produce the target proteins with increased purity and homogeneity, which would render them more likely to yield well diffracting crystals. This is performed by alternative purification procedures and/or the introduction of chemical modifications to the proteins (such as tag removal, methylation, surface mutagenesis, selenomethionine labelling etc.). Details of the various procedures in the pipeline for protein crystallization, target salvaging, data collection/processing and high-throughput structure determination/validation, as well as some examples, are described.
Assuntos
Cristalografia por Raios X/métodos , Proteínas/química , Cristalização , Proteínas/isolamento & purificaçãoRESUMO
Recently, the demands of high-throughput macromolecular crystallography have driven continuous improvements in phasing methods, data-collection protocols and many other technologies. Single-wavelength anomalous scattering (SAS) phasing with chromium X-ray radiation opens a new possibility for phasing a protein with data collected in-house and has led to several successful examples of de novo structure solution using only weak anomalous scatterers such as sulfur. To further reduce data-collection time and make SAS phasing more robust, it is natural to combine selenomethionine-derivatized protein (SeMet protein) with Cr Kalpha radiation to take advantage of the larger anomalous scattering signal from selenium (f'' = 2.28 e(-)) compared with sulfur (f'' = 1.14 e(-)). As reported herein, the crystal structure of a putative chorismate mutase from Clostridium thermocellum was determined using Se-SAS with Cr Kalpha radiation. Each protein molecule contains eight selenomethionine residues in 148 amino-acid residues, providing a calculated Bijvoet ratio of about 3.5% at the Cr Kalpha wavelength. A single data set to 2.2 A resolution with approximately ninefold redundancy was collected using an imaging-plate detector coupled with a Cr source. Structure solution, refinement and deposition to the Protein Data Bank were performed within 9 h of the availability of the scaled diffraction data. The procedure used here is applicable to many other proteins and promises to become a routine pathway for in-house high-throughput crystallography.
Assuntos
Corismato Mutase/química , Cromo/química , Cristalografia por Raios X/métodos , Proteínas/química , Clostridium thermocellum/enzimologia , Estrutura Secundária de Proteína , Espalhamento de Radiação , Selenoproteínas , Difração de Raios XRESUMO
Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2,200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.