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Antibiotics are the mainstay of therapy for bacterial vaginosis (BV). However, the rate of treatment failure in patients with recurrent BV is about 50%. Herein, we investigated potential mechanisms of therapy failure, including the propensity of resistance formation and biofilm activity of metronidazole (MDZ), clindamycin (CLI), and PM-477, a novel investigational candidate that is a genetically engineered endolysin with specificity for bacteria of the genus Gardnerella. Determination of the MIC indicated that 60% of a panel of 22 Gardnerella isolates of four different species were resistant to MDZ, while all strains were highly susceptible to CLI and to the endolysin PM-477. Six strains, all of which were initially susceptible to MDZ, were passaged with MDZ or its more potent hydroxy metabolite. All of them generated full resistance after 5 to 10 passages, resulting in MICs of >512 µg/mL. In contrast, only a mild increase in MIC was found for PM-477. There was also no cross-resistance formation, as MDZ-resistant Gardnerella strains remained highly susceptible to PM-477, both in suspension and in preformed biofilms. Strains that were resistant to MDZ in suspension were also tolerant to MDZ at >2,048 µg/mL when growing as biofilm. All strains were susceptible to PM-477 when grown as preformed biofilms, at minimum biofilm eradication concentrations (MBECs) in the range of 1 to 4 µg/mL. Surprisingly, the MBEC of CLI was >512 µg/mL for 7 out of 9 tested Gardnerella strains, all of which were susceptible to CLI when growing in suspension. The observed challenges of MDZ and CLI due to resistance formation and ineffectiveness on biofilm, respectively, could be one explanation for the frequent treatment failures in uncomplicated or recurrent BV. Therefore, the high efficacy of PM-477 in eliminating Gardnerella in in vitro biofilms, as well as its high resilience to resistance formation, makes PM-477 a promising potential alternative for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.
Assuntos
Vaginose Bacteriana , Biofilmes , Clindamicina/farmacologia , Clindamicina/uso terapêutico , Endopeptidases , Feminino , Gardnerella , Gardnerella vaginalis , Humanos , Metronidazol/uso terapêutico , Vaginose Bacteriana/tratamento farmacológico , Vaginose Bacteriana/microbiologiaRESUMO
The HIV-1 protein Rev is essential for virus replication and ensures the expression of partially spliced and unspliced transcripts. We identified a ULM (UHM ligand motif) motif in the Arginine-Rich Motif (ARM) of the Rev protein. ULMs (UHM ligand motif) mediate protein interactions during spliceosome assembly by binding to UHM (U2AF homology motifs) domains. Using NMR, biophysical methods and crystallography we show that the Rev ULM binds to the UHMs of U2AF65 and SPF45. The highly conserved Trp45 in the Rev ULM is crucial for UHM binding in vitro, for Rev co-precipitation with U2AF65 in human cells and for proper processing of HIV transcripts. Thus, Rev-ULM interactions with UHM splicing factors contribute to the regulation of HIV-1 transcript processing, also at the splicing level. The Rev ULM is an example of viral mimicry of host short linear motifs that enables the virus to interfere with the host molecular machinery.
Assuntos
Infecções por HIV/genética , HIV-1/genética , Fator de Processamento U2AF/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Processamento Alternativo/genética , Motivos de Aminoácidos/genética , Arginina/genética , Regulação Viral da Expressão Gênica/genética , Infecções por HIV/virologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Ligação Proteica/genética , Fatores de Processamento de RNA/genética , Spliceossomos/genética , Replicação Viral/genéticaRESUMO
S. aureus is a pathogen that frequently causes severe morbidity and phage therapy is being discussed as an alternative to antibiotics for the treatment of S. aureus infections. In this in vitro and animal study, we demonstrated that the activity of anti-staphylococcal phages is severely impaired in 0.5% plasma or synovial fluid. Despite phage replication in these matrices, lysis of the bacteria was slower than phage propagation, and no reduction of the bacterial population was observed. The inhibition of the phages associated with a reduction in phage adsorption, quantified to 99% at 10% plasma. S. aureus is known to bind multiple coagulation factors, resulting in the formation of aggregates and blood clots that might protect the bacterium from the phages. Here, we show that purified fibrinogen at a sub-physiological concentration of 0.4 mg/ml is sufficient to impair phage activity. In contrast, dissolution of the clots by tissue plasminogen activator (tPA) partially restored phage activity. Consistent with these in vitro findings, phage treatment did not reduce bacterial burdens in a neutropenic mouse S. aureus thigh infection model. In summary, phage treatment of S. aureus infections inside the body may be fundamentally challenging, and more investigation is needed prior to proceeding to in-human trials.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Animais , Camundongos , Staphylococcus aureus/fisiologia , Ativador de Plasminogênio Tecidual , Líquido Sinovial , Infecções Estafilocócicas/terapia , Infecções Estafilocócicas/microbiologia , Fagos de Staphylococcus/fisiologia , AntibacterianosRESUMO
We present the mass balances associated with carbon dioxide (CO2) removal (CDR) using seawater as both the source of reactants and as the reaction medium via electrolysis following the "Equatic" (formerly known as "SeaChange") process. This process, extensively detailed in La Plante E.C.; ACS Sustain. Chem. Eng.2021, 9, ( (3), ), 1073-1089, involves the application of an electric overpotential that splits water to form H+ and OH- ions, producing acidity and alkalinity, i.e., in addition to gaseous coproducts, at the anode and cathode, respectively. The alkalinity that results, i.e., via the "continuous electrolytic pH pump" results in the instantaneous precipitation of calcium carbonate (CaCO3), hydrated magnesium carbonates (e.g., nesquehonite: MgCO3·3H2O, hydromagnesite: Mg5(CO3)4(OH)2·4H2O, etc.), and/or magnesium hydroxide (Mg(OH)2) depending on the CO32- ion-activity in solution. This results in the trapping and, hence, durable and permanent (at least â¼10â¯000-100â¯000 years) immobilization of CO2 that was originally dissolved in water, and that is additionally drawn down from the atmosphere within: (a) mineral carbonates, and/or (b) as solvated bicarbonate (HCO3-) and carbonate (CO32-) ions (i.e., due to the absorption of atmospheric CO2 into seawater having enhanced alkalinity). Taken together, these actions result in the net removal of â¼4.6 kg of CO2 per m3 of seawater catholyte processed. Geochemical simulations quantify the extents of net CO2 removal including the dependencies on the process configuration. It is furthermore indicated that the efficiency of realkalinization of the acidic anolyte using alkaline solids depends on their acid neutralization capacity and dissolution reactivity. We also assess changes in seawater chemistry resulting from Mg(OH)2 dissolution with emphasis on the change in seawater alkalinity and saturation state. Overall, this analysis provides direct quantifications of the ability of the Equatic process to serve as a means for technological CDR to mitigate the worst effects of accelerating climate change.
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The U2AF-homology motif (UHM) mediates protein-protein interactions between factors involved in constitutive RNA splicing. Here we report that the splicing factor SPF45 regulates alternative splicing of the apoptosis regulatory gene FAS (also called CD95). The SPF45 UHM is necessary for this activity and binds UHM-ligand motifs (ULMs) present in the 3' splice site-recognizing factors U2AF65, SF1 and SF3b155. We describe a 2.1-A crystal structure of SPF45-UHM in complex with a ULM peptide from SF3b155. Features distinct from those of previously described UHM-ULM structures allowed the design of mutations in the SPF45 UHM that selectively impair binding to individual ULMs. Splicing assays using the ULM-selective SPF45 variants demonstrate that individual UHM-ULM interactions are required for FAS splicing regulation by SPF45 in vivo. Our data suggest that networks of UHM-ULM interactions are involved in regulating alternative splicing.
Assuntos
Processamento Alternativo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Receptor fas/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Éxons , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/química , Peptídeos/química , Fosfoproteínas/química , Conformação Proteica , Mapeamento de Interação de Proteínas , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteínas/química , Fator de Processamento U2AFRESUMO
Bacterial vaginosis (BV) is the most frequent vaginal infection in women of reproductive age. It is caused by the overgrowth of anaerobic vaginal pathogens, such as Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia, which are vaginal pathogens detected during the early stages of incident BV and have been found to form multi-species biofilms. Treatment of biofilm-associated infections, such as BV, is challenging. In this study, we tested the role of an investigational engineered phage endolysin, PM-477, in the eradication of dual-species biofilms composed of G. vaginalis-F. vaginae or G. vaginalis-P. bivia. Single-species biofilms formed by these species were also analysed as controls. The effect of PM-477 on biomass and culturability of single- and dual-species biofilms was assessed in vitro using a microtiter plate assay, epifluorescence microscopy, confocal laser scanning microscopy, and quantitative PCR. The results showed that PM-477 was particularly effective in the disruption and reduction of culturability of G. vaginalis biofilms. In dual-species biofilms, PM-477 exhibited lower efficiency but was still able to selectively and significantly eliminate G. vaginalis. Since polymicrobial interactions have been shown to strongly affect the activity of various antibiotics, the activity of PM-477 in dual-species biofilms is a potentially promising result that should be further explored, aiming to completely eradicate multi-species biofilms associated with BV.
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BACKGROUND: Testing of antibiotic resistance of intact vaginal microbiota in pure culture is not feasible. METHODS: Metronidazole, antiseptic octenisept®, antimycotic ciclopirox, bacterial probiotic Lactobacillus crispatus, yeast probiotic Saccharomyces boulardii, Gardnerella-phage-endolysin named phagolysin and phagolysin in combination with probiotics were tested for bacteriolytic activity. Included were vaginal swabs from 38 random women with Amsel-confirmed bacterial vaginosis (BV). Test aliquots were incubated by 37° for 2 and 24 h. Gardnerella, low G+C, Atopobium, lactobacilli, Lactobacillus iners and crispatus, Prevotella-Bacteroides, and Gammaproteobacteria microbial groups were quantified using fluorescence in situ hybridization (FISH). RESULTS: The probiotic strain Lactobacillus crispatus demonstrated the weakest bacteriolytical effects, followed by metronidazole. Both had no impact on Gardnerella species, instead lysing Prevotella-Bacteroides, Enterobacteriaceae (by L.crispatus) or LGC, Atopobium and Prevotella-Bacteroides (by metronidazole) groups of the microbiota. Cytolytic activity on Gardnerella was highly pronounced and increased from octenisept to ciclopirox, phagolysin, phagolysin with L.crispatus, being best in the combination of phagolysin with S.boulardii. Universally active ciclopirox and octenisept® suppressed nearly all microbial groups including those which are regarded as beneficial. Phagolysin had no effect on naturally occurring Lactobacillus crispatus. Conclusions: FISH susceptibility testing allows unique efficacy evaluation of individually adjusted topical therapy without microbial isolation facilitating optimal therapy choice.
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Quantification of the number of living cells in biofilm or after eradication treatments of biofilm, is problematic for different reasons. We assessed the performance of pre-treatment of DNA, planktonic cells and ex vivo vaginal biofilms of Gardnerella with propidium monoazide (PMAxx) to prevent qPCR-based amplification of DNA from killed cells (viability-qPCR). Standard PMAxx treatment did not completely inactivate free DNA and did not affect living cells. While culture indicated that killing of planktonic cells by heat or by endolysin was complete, viability-qPCR assessed only log reductions of 1.73 and 0.32, respectively. Therefore, we improved the standard protocol by comparing different (combinations of) parameters, such as concentration of PMAxx, and repetition, duration and incubation conditions of treatment. The optimized PMAxx treatment condition for further experiments consisted of three cycles, each of: 15 min incubation on ice with 50 µM PMAxx, followed by 15 min-long light exposure. This protocol was validated for use in vaginal samples from women with bacterial vaginosis. Up to log2.2 reduction of Gardnerella cells after treatment with PM-477 was documented, despite the complex composition of the samples, which might have hampered the activity of PM-477 as well as the quantification of low loads by viability-qPCR.
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Here, we present a case of a 79-year-old female with a recalcitrant Staphylococcal epidermidis prosthetic knee infection that was successfully treated with a single dose of adjuvant intra-articular bacteriophage therapy after debridement and implant retention surgery. The bacteriophage used in this case, PM448, is the first É2 bacteriophage to be used in vivo. Currently the patient is without evidence of clinical recurrence and, interestingly, the patient had also suffered from debilitating aplastic anemia for over 2 years, which is recovering since receiving adjuvant bacteriophage therapy.
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Alternative treatments for Escherichia coli infections are urgently needed, and phage therapy is a promising option where antibiotics fail, especially for urinary tract infections (UTI). We used wastewater-isolated phages to test their lytic activity against a panel of 47 E. coli strains reflecting the diversity of strains found in UTI, including sequence type 131, 73 and 69. The plaquing host range (PHR) was between 13 and 63%. In contrast, the kinetic host range (KHR), describing the percentage of strains for which growth in suspension was suppressed for 24 h, was between 0% and 19%, substantially lower than the PHR. To improve the phage host range and their efficacy, we bred the phages by mixing and propagating cocktails on a subset of E. coli strains. The bred phages, which we termed evolution-squared ε2-phages, of a mixture of Myoviridae have KHRs up to 23% broader compared to their ancestors. Furthermore, using constant phage concentrations, Myoviridae ε2-phages suppressed the growth of higher bacterial inocula than their ancestors did. Thus, the ε2-phages were more virulent compared to their ancestors. Analysis of the genetic sequences of the ε2-phages with the broadest host range reveals that they are mosaic intercrossings of 2-3 ancestor phages. The recombination sites are distributed over the whole length of the genome. All ε2-phages are devoid of genes conferring lysogeny, antibiotic resistance, or virulence. Overall, this study shows that ε2-phages are remarkably more suitable than the wild-type phages for phage therapy.
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Due to the rapid spread of antibiotic resistance, and the difficulties of treating biofilm-associated infections, alternative treatments for S. aureus infections are urgently needed. We tested the lytic activity of several wild type phages against a panel of 110 S. aureus strains (MRSA/MSSA) composed to reflect the prevalence of S. aureus clonal complexes in human infections. The plaquing host ranges (PHR) of the wild type phages were in the range of 51% to 60%. We also measured what we called the kinetic host range (KHR), i.e., the percentage of strains for which growth in suspension was suppressed for 24 h. The KHR of the wild type phages ranged from 2% to 49%, substantially lower than the PHRs. To improve the KHR and other key pharmaceutical properties, we bred the phages by mixing and propagating cocktails on a subset of S. aureus strains. These bred phages, which we termed evolution-squared (ε2) phages, have broader KHRs up to 64% and increased virulence compared to the ancestors. The ε2-phages with the broadest KHR have genomes intercrossed from up to three different ancestors. We composed a cocktail of three ε2-phages with an overall KHR of 92% and PHR of 96% on 110 S. aureus strains and called it PM-399. PM-399 has a lower propensity to resistance formation than the standard of care antibiotics vancomycin, rifampicin, or their combination, and no resistance was observed in laboratory settings (detection limit: 1 cell in 1011). In summary, ε2-phages and, in particular PM-399, are promising candidates for an alternative treatment of S. aureus infections.
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Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13-8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.
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To be successful, academic and commercial efforts to reintroduce phage therapy must ensure that only safe and efficacious products are used to treat patients. This raises a number of manufacturing, formulation, and delivery challenges. Since phages are biologics, robust manufacturing processes will be crucial to avoid unwanted variability in each step of the process. The quality standards themselves need to be developed, as patients are currently being treated with phages produced under quality standards ranging from cGMP for clinical trials in EMA and FDA regulated environments to no standards at all in some last resort treatments. In this short review, we will systematically review the literature covering technical issues and approaches to increase robustness at every step of the production process: the identity of the phage and bacterial production strains, the fermentation process and purification, the formulation of the drug product, the quality controls and the documentation standards themselves. We conclude that it is possible to control cost at the same time, which is critical to re-introduce phage therapy to western medicine.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Lisina/metabolismo , Metilação , Camundongos , Proteínas Nucleares/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Schizosaccharomyces pombe/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
The role of a protein inside a cell is determined by both its location and its conformational state. Although fluorescence techniques are widely used to determine the cellular localization of proteins in vivo, these approaches cannot provide detailed information about a protein's three-dimensional state. This gap, however, can be filled by NMR spectroscopy, which can be used to investigate both the conformation as well as the dynamics of proteins inside living cells. In this chapter we describe technical aspects of these "in-cell NMR" experiments. In particular, we show that in the case of (15)N-labeling schemes the background caused by labeling all cellular components is negligible, while (13)C-based experiments suffer from high background levels and require selective labeling schemes. A correlation between the signal-to-noise ratio of in-cell NMR experiments with the overexpression level of the protein shows that the current detection limit is 150-200 muM (intracellular concentration). We also discuss experiments that demonstrate that the intracellular viscosity is not a limiting factor since the intracellular rotational correlation time is only approximately two times longer than the correlation time in water. Furthermore, we describe applications of the technique and discuss its limitations.
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Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Calmodulina/química , Calmodulina/metabolismo , Escherichia coli/metabolismo , Técnicas In Vitro , Isótopos de Nitrogênio , Proteínas/metabolismoRESUMO
Classic nuclear export signals (NESs) confer CRM1-dependent nuclear export. Here we present crystal structures of the RanGTP-CRM1 complex alone and bound to the prototypic PKI or HIV-1 Rev NESs. These NESs differ markedly in the spacing of their key hydrophobic (Φ) residues, yet CRM1 recognizes them with the same rigid set of five Φ pockets. The different Φ spacings are compensated for by different conformations of the bound NESs: in the case of PKI, an α-helical conformation, and in the case of Rev, an extended conformation with a critical proline docking into a Φ pocket. NMR analyses of CRM1-bound and CRM1-free PKI NES suggest that CRM1 selects NES conformers that pre-exist in solution. Our data lead to a new structure-based NES consensus, and explain why NESs differ in their affinities for CRM1 and why supraphysiological NESs bind the exportin so tightly.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Carioferinas/química , Sinais de Exportação Nuclear , Receptores Citoplasmáticos e Nucleares/química , Proteína ran de Ligação ao GTP/química , Sítios de Ligação , Sequência Consenso , Cristalografia por Raios X , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Carioferinas/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Mutação Puntual , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Proteína Exportina 1RESUMO
The Tudor-SN protein (p100, SND1) has been implicated in a variety of cellular processes, such as transcription, processing of edited double-stranded RNA, and splicing regulation. Molecular details of these functions are not yet understood. Tudor domains have previously been shown to bind methylated ligands, such as methylated lysines and arginines. It has been suggested that the role of Tudor-SN in splicing may involve binding to such methylated ligands or to the methylated 5' cap of spliceosomal snRNAs. Here, we report the crystal structure of the extended Tudor domain of Tudor-SN from Drosophila melanogaster to a resolution of 2.1 A. NMR secondary chemical shifts, relaxation data, and residual dipolar couplings indicate that the solution and crystal structures are similar. Binding of various ligands was investigated by NMR. Binding sites and affinities were characterized by chemical shift perturbations. We show that the aromatic cage of the Tudor domain specifically binds a peptide containing symmetrically dimethylated arginines (sDMA) with micromolar affinity, while the same peptide comprising nonmethylated arginines does not show significant chemical shift perturbations. Tudor-SN preferentially recognizes sDMA over asymmetrically dimethylated arginine (aDMA). In contrast, two 5' cap analogues with different methylation patterns, as well as mono-, di-, and trimethyllysines, show no binding. Our data demonstrate that the Tudor domain of Tudor-SN specifically recognizes sDMA-containing ligands. The aromatic cage of Tudor-SN is very similar to the one in the Tudor domain of the survival of motor neuron protein, which also recognizes sDMA peptides, indicating a conserved binding motif for this methylation mark. Recognition of sDMA in the C-terminal tails of spliceosomal Sm proteins suggests how Tudor-SN may interact with small nuclear ribonucleoprotein particles during the regulation of splicing.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Sequência de Aminoácidos , Animais , Arginina/análogos & derivados , Arginina/química , Arginina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endonucleases , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Eletricidade Estática , TermodinâmicaRESUMO
PUF60 is an essential splicing factor functionally related and homologous to U2AF(65). Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophan-containing linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 angstroms resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF65, and SF3b155. Compared with U2AF65-UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.
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Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Fosfoproteínas/química , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteínas/química , Fatores de Transcrição/química , Motivos de Aminoácidos/fisiologia , Animais , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA/metabolismo , Dimerização , Humanos , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Fatores de Processamento de RNA , Proteínas de Ligação a RNA , Proteínas Repressoras , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteínas/metabolismo , Homologia de Sequência de Aminoácidos , Dodecilsulfato de Sódio/química , Fator de Processamento U2AF , Fatores de Transcrição/metabolismoRESUMO
U2AF homology motifs (UHM) are protein domains that bind peptidic UHM ligand motifs (ULM) and thus form an intricate network of interactions involved in splicing regulation. Here, we report the backbone assignment of the UHM domain of the splicing factor Puf60 as well as (1)H, (15)N chemical shifts upon binding of the ULM peptides U2AF(65) (85-112), SF1 (1-25), SF3b155 (194-229), SF3b155 (317-357), and Prp16 (201-238).
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Proteínas de Transporte/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas Nucleares , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteínas , Sequência de Aminoácidos , Isótopos de Carbono/química , Dados de Sequência Molecular , Peso Molecular , Isótopos de Nitrogênio/química , Estrutura Terciária de Proteína , Prótons , Fator de Processamento U2AFRESUMO
Structural investigations are frequently hindered by difficulties in obtaining diffracting crystals of the target protein. Here, we report the crystallization and structure solution of the U2AF homology motif (UHM) domain of splicing factor Puf60 fused to Escherichia coli thioredoxin A. Both modules make extensive crystallographic contacts, contributing to a well-defined crystal lattice with clear electron density for both the thioredoxin and the Puf60-UHM module. We compare two short linker sequences between the two fusion domains, GSAM and GSPPM, for which only the GSAM-linked fusion protein yielded diffracting crystals. While specific interdomain contacts are not observed for both fusion proteins, NMR relaxation data in solution indicate reduced interdomain mobility between the Trx and Puf60-UHM modules. The GSPPM-linked fusion protein is significantly more flexible, albeit both linker sequences have the same number of degrees of torsional freedom. Our analysis provides a rationale for the crystallization of the GSAM-linked fusion protein and indicates that in this case, a four-residue linker between thioredoxin A and the fused target may represent the maximal length for crystallization purposes. Our data provide an experimental basis for the rational design of linker sequences in carrier-driven crystallization and identify thioredoxin A as a powerful fusion partner that can aid crystallization of difficult targets.