RESUMO
We unveil the intimate relationship between protein dynamics and allostery by following the trajectories of model proteins in their conformational and sequence spaces. Starting from a nonallosteric hyperthermophilic malate dehydrogenase, we have tracked the role of protein dynamics in the evolution of the allosteric capacity. Based on a large phylogenetic analysis of the malate (MalDH) and lactate dehydrogenase (LDH) superfamily, we identified two amino acid positions that could have had a major role for the emergence of allostery in LDHs, which we targeted for investigation by site-directed mutagenesis. Wild-type MalDH and the single and double mutants were tested with respect to their substrate recognition profiles. The double mutant displayed a sigmoid-shaped profile typical of homotropic activation in LDH. By using molecular dynamics simulations, we showed that the mutations induce a drastic change in the protein sampling of its conformational landscape, making transiently T-like (inactive) conformers, typical of allosteric LDHs, accessible. Our data fit well with the seminal key concept linking protein dynamics and evolvability. We showed that the selection of a new phenotype can be achieved by a few key dynamics-enhancing mutations causing the enrichment of low-populated conformational substates.
Assuntos
Malato Desidrogenase , Malatos , Regulação Alostérica , Aminoácidos/genética , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/genética , Mutação , FilogeniaRESUMO
Recombinant adeno-associated virus virus-derived vectors (rAAVs) are among the most used viral delivery system for in vivo gene therapies with a good safety profile. However, rAAV production methods often lead to a heterogeneous vector population, in particular with the presence of undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is considered as the gold analytical technique allowing the measurement of relative amounts of each vector subpopulation and components like particle aggregates, based on their sedimentation coefficients. This letter presents the principle and practice of AUC experiments for rAAVs characterization. We discuss our results in the framework of previously published works. In addition to classical detection at 260 nm, using interference optics in the ultracentrifuge can provide an independent estimate of weight percentages of the different populations of capsids, and of the genome size incorporated in rAAV particles.
Assuntos
Dependovirus , Vetores Genéticos , Dependovirus/genética , Terapia Genética , Ultracentrifugação/métodosRESUMO
Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the ß-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.
Assuntos
Proteínas de Bactérias , Esporos Bacterianos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Estrutura Secundária de Proteína , Esporos Bacterianos/metabolismoRESUMO
S100A9, with its congener S100A8, belongs to the S100 family of calcium-binding proteins found exclusively in vertebrates. These two proteins are major constituents of neutrophils. In response to a pathological condition, they can be released extracellularly and become alarmins that induce both pro- and anti-inflammatory signals, through specific cell surface receptors. They also act as antimicrobial agents, mainly as a S100A8/A9 heterocomplex, through metal sequestration. The mechanisms whereby divalent cations modulate the extracellular functions of S100A8 and S100A9 are still unclear. Importantly, it has been proposed that these ions may affect both the ternary and quaternary structure of these proteins, thereby influencing their physiological properties. In the present study, we report the crystal structures of WT and C80A murine S100A9 (mS100A9), determined at 1.45 and 2.35 Å resolution, respectively, in the presence of calcium and zinc. These structures reveal a canonical homodimeric form for the protein. They also unravel an intramolecular disulfide bridge that stabilizes the C-terminal tail in a rigid conformation, thus shaping a second Zn-binding site per S100A9 protomer. In solution, mS100A9 apparently binds only two zinc ions per homodimer, with an affinity in the micromolar range, and aggregates in the presence of excess zinc. Using mass spectrometry, we demonstrate that mS100A9 can form both non-covalent and covalent homodimers with distinct disulfide bond patterns. Interestingly, calcium and zinc seem to affect differentially the relative proportion of these forms. We discuss how the metal-dependent interconversion between mS100A9 homodimers may explain the versatility of physiological functions attributed to the protein.
Assuntos
Calgranulina B/metabolismo , Cátions Bivalentes/metabolismo , Dissulfetos/metabolismo , Animais , Sítios de Ligação/fisiologia , Cálcio/metabolismo , Dimerização , Camundongos , Domínios Proteicos/fisiologia , Zinco/metabolismoRESUMO
Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.
Assuntos
Benchmarking , Laboratórios , Calorimetria , Reprodutibilidade dos Testes , TemperaturaRESUMO
The structural characterization of modular proteins containing long intrinsically disordered regions intercalated with folded domains is complicated by their conformational diversity and flexibility and requires the integration of multiple experimental approaches. Nipah virus (NiV) phosphoprotein, an essential component of the viral RNA transcription/replication machine and a component of the viral arsenal that hijacks cellular components and counteracts host immune responses, is a prototypical model for such modular proteins. Curiously, the phosphoprotein of NiV is significantly longer than the corresponding protein of other paramyxoviruses. Here, we combine multiple biophysical methods, including x-ray crystallography, NMR spectroscopy, and small angle x-ray scattering, to characterize the structure of this protein and provide an atomistic representation of the full-length protein in the form of a conformational ensemble. We show that full-length NiV phosphoprotein is tetrameric, and we solve the crystal structure of its tetramerization domain. Using NMR spectroscopy and small angle x-ray scattering, we show that the long N-terminal intrinsically disordered region and the linker connecting the tetramerization domain to the C-terminal X domain exchange between multiple conformations while containing short regions of residual secondary structure. Some of these transient helices are known to interact with partners, whereas others represent putative binding sites for yet unidentified proteins. Finally, using NMR spectroscopy and isothermal titration calorimetry, we map a region of the phosphoprotein, comprising residues between 110 and 140 and common to the V and W proteins, that binds with weak affinity to STAT1 and confirm the involvement of key amino acids of the viral protein in this interaction. This provides new, to our knowledge, insights into how the phosphoprotein and the nonstructural V and W proteins of NiV perform their multiple functions.
Assuntos
Vírus Nipah , Fosfoproteínas , Conformação Proteica , Proteínas Virais , Replicação ViralRESUMO
BACKGROUND: Our previous studies showed increased angiotensin I-converting enzyme (ACE) activity in chronic schizophrenia (SCZ) patients compared to healthy control (HC) volunteers, and the relevance of combining ACE genotype and activity for predicting SCZ was suggested. METHODS: ACE activity was measured in plasma of ACE insertion/deletion (I/D) genotyped HC volunteers (N = 53) and antipsychotic-naïve first-episode psychosis (FEP) patients (N = 45), assessed at baseline (FEB-B) and also after 2-months (FEP-2M) of treatment with the atypical antipsychotic risperidone. RESULTS: ACE activity measurements showed significant differences among HC, FEP-B and FEP-2M groups (F = 5.356, df = 2, p = 0.005), as well as between HC and FEP-2M (post-hoc Tukey's multiple comparisons test, p = 0.004). No correlation was observed for ACE activity increases and symptom severity reductions in FEP as assessed by total PANSS (r = -0.131, p = 0.434). FEP subgrouped by ACE I/D genotype showed significant ACE activity increases, mainly in the DD genotype subgroup. No correlation between ACE activity and age was observed in FEP or HC groups separately (r = 0.210, p = 0.392), but ACE activity levels differences observed between these groups were influenced by age. CONCLUSIONS: The importance of measuring the ACE activity in blood plasma, associated to ACE I/D genotyping to support the follow-up of FEP patients did not show correlation with general symptoms amelioration in the present study. However, new insights into the influence of age and I/D genotype for ACE activity changes in FEP individuals upon treatment was demonstrated.
RESUMO
Approximately 255 million people consume illicit drugs every year, among which 18 million use cocaine. A portion of this drug is represented by crack, but it is difficult to estimate the number of users since most are marginalized. However, there are no recognized efficacious pharmacotherapies for crack-cocaine dependence. Inflammation and infection in cocaine users may be due to behavior adopted in conjunction with drug-related changes in the brain. To understand the metabolic changes associated with the drug abuse disorder and identify biomarkers, we performed a 1H NMR-based metabonomic analysis of 44 crack users' and 44 healthy volunteers' blood serum. The LDA model achieved 98% of accuracy. From the water suppressed 1H NMR spectra analyses, it was observed that the relative concentration of lactate was higher in the crack group, while long chain fatty acid acylated carnitines were decreased, which was associated with their nutritional behavior. Analyses of the aromatic region of CPMG 1H NMR spectra demonstrated histidine and tyrosine levels increased in the blood serum of crack users. The reduction of carnitine and acylcarnitines and the accumulation of histidine in the serum of the crack users suggest that histamine biosynthesis is compromised. The tyrosine level points to altered dopamine concentration.
Assuntos
Transtornos Relacionados ao Uso de Cocaína , Cocaína Crack/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Metaboloma/efeitos dos fármacos , Coleta de Amostras Sanguíneas , Carnitina/sangue , Estudos de Casos e Controles , Histidina/sangue , Humanos , Ácido Láctico/sangue , Tirosina/sangueRESUMO
The NAD(P)-dependent malate dehydrogenases (MalDHs) and NAD-dependent lactate dehydrogenases (LDHs) are homologous enzymes involved in central metabolism. They display a common protein fold and the same catalytic mechanism, yet have a stringent capacity to discriminate between their respective substrates. The MalDH/LDH superfamily is divided into several phylogenetically related groups. It has been shown that the canonical LDHs and LDH-like group of MalDHs are primarily tetrameric enzymes that diverged from a common ancestor. In order to gain understanding of the evolutionary history of the LDHs and MalDHs, the biochemical properties and crystallographic structure of the LDH-like MalDH from the hyperthermophilic archaeon Ignicoccus islandicus (I. isl) were determined. I. isl MalDH recognizes oxaloacetate as main substrate, but it is also able to use pyruvate. Surprisingly, with pyruvate, the enzymatic activity profile looks like that of allosteric LDHs, suggesting a hidden allosteric capacity in a MalDH. The I. isl MalDH tetrameric structure in the apo state is considerably different from those of canonical LDH-like MalDHs and LDHs, representing an alternative oligomeric organization. A comparison with MalDH and LDH counterparts provides strong evidence that the divergence between allosteric and non-allosteric members of the superfamily involves homologs with intermediate, atypical properties.
Assuntos
Archaea/enzimologia , Proteínas Arqueais/metabolismo , Malato Desidrogenase/metabolismo , Proteínas Arqueais/química , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/química , Multimerização Proteica , Especificidade por SubstratoRESUMO
The Gram-positive bacterium Bacillus subtilis responds to starvation by entering a morphological differentiation process leading to the formation of a highly resistant spore. Early in the sporulation process, the cell asymmetrically divides into a large compartment (the mother cell) and a smaller one (the forespore), which will maturate into a resistant spore. Proper development of the forespore requires the assembly of a multiprotein complex called the SpoIIIA-SpoIIQ complex or "A-Q complex". This complex involves the forespore protein SpoIIQ and eight mother cell proteins (SpoIIIAA to SpoIIIAH), many of which share structural similarities with components of specialized secretion systems and flagella found in Gram-negative bacteria. The assembly of the A-Q complex across the two membranes that separate the mother cell and forespore was recently shown to require GerM. GerM is a lipoprotein composed of two GerMN domains, a family of domains with unknown function. Here, we report X-ray crystallographic structures of the first GerMN domain of GerM at 1.0â¯Å resolution, and of the soluble domain of GerM (the tandem of GerMN domains) at 2.1â¯Å resolution. These structures reveal that GerMN domains can adopt distinct conformations and that the core of these domains display structural similarities with ring-building motifs found in components of specialized secretion system and in SpoIIIA proteins. This work provides an additional piece towards the structural characterization of the A-Q complex.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Hidrolases/metabolismo , Esporos Bacterianos/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Hidrolases/química , Hidrolases/genética , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Homologia de Sequência de AminoácidosRESUMO
The efficacy of crotamine as antitumoral was first demonstrated by daily intraperitoneal (IP) injections of low doses of this toxin in an animal model bearing melanoma tumors. Significant inhibition of tumor growth and increased lifespan of mice bearing tumor was also noticed after 21 consecutive days of this daily IP administration of crotamine. However, due to the limited acceptance of treatments by IP route in clinical conditions, herein, we evaluated the antitumor effect of this native polypeptide employing the oral route. The efficacy of crotamine in inhibiting the melanoma growth in vivo, even after passing through the gastrointestinal tract of the animal, was confirmed here. In addition, biochemical biomarkers and also histopathological analysis showed both the absence of any potential toxic effects in tissues or organs of the animal in which the highest accumulation of crotamine is expected. Interestingly, a reduction of weight gain was observed mainly in animals with tumor treated with crotamine by IP route, but not by oral administration. Albeit, oral administered crotamine was able to significantly decrease the body weight gain of healthy animals without tumor. Taking advantage of this same experimental animal models receiving crotamine by oral route, it was possible to show metabolic changes as the increased capacity of glucose clearance, which was accompanied by a reduction of the total cholesterol, and by increased high-density lipoprotein levels, both observed mainly in the absence of tumor. Triglycerides and low-density lipoprotein were also significantly decreased, but only in the absence of tumor. Taken together, these data suggest a clear trend for metabolic positive effects and mischaracterize unhealthy condition of animals, with or without tumors, treated with crotamine for 21 days. In addition, this study confirmed the efficacy of crotamine administered by oral route as antitumor agent, which besides the additional advantage of administration convenience and decreased risk of toxic effects, allowed the serendipitous observation of several positive metabolic effects on treated animals.
Assuntos
Venenos de Crotalídeos/administração & dosagem , Venenos de Crotalídeos/farmacologia , Melanoma Experimental/tratamento farmacológico , Metaboloma/efeitos dos fármacos , Venenos de Serpentes/química , Administração Oral , Sequência de Aminoácidos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Venenos de Crotalídeos/toxicidade , Crotalus , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Aumento de Peso/efeitos dos fármacosRESUMO
The sorting nexin 27 (SNX27)-retromer complex is a major regulator of endosome-to-plasma membrane recycling of transmembrane cargos that contain a PSD95, Dlg1, zo-1 (PDZ)-binding motif. Here we describe the core interaction in SNX27-retromer assembly and its functional relevance for cargo sorting. Crystal structures and NMR experiments reveal that an exposed ß-hairpin in the SNX27 PDZ domain engages a groove in the arrestin-like structure of the vacuolar protein sorting 26A (VPS26A) retromer subunit. The structure establishes how the SNX27 PDZ domain simultaneously binds PDZ-binding motifs and retromer-associated VPS26. Importantly, VPS26A binding increases the affinity of the SNX27 PDZ domain for PDZ- binding motifs by an order of magnitude, revealing cooperativity in cargo selection. With disruption of SNX27 and retromer function linked to synaptic dysfunction and neurodegenerative disease, our work provides the first step, to our knowledge, in the molecular description of this important sorting complex, and more broadly describes a unique interaction between a PDZ domain and an arrestin-like fold.
Assuntos
Endocitose/fisiologia , Domínios PDZ/genética , Nexinas de Classificação/química , Proteínas de Transporte Vesicular/química , Sequência de Aminoácidos , Animais , Arrestina/química , Arrestina/genética , Encefalopatias/genética , Encefalopatias/metabolismo , Encefalopatias/patologia , Cristalografia por Raios X , Endossomos/metabolismo , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Dobramento de Proteína , Sinais Direcionadores de Proteínas/genética , RNA Interferente Pequeno/genética , Ratos , Homologia de Sequência de Aminoácidos , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Sorting nexins (SNXs) or phox homology (PX) domain containing proteins are central regulators of cell trafficking and signaling. A subfamily of PX domain proteins possesses two unique PX-associated domains, as well as a regulator of G protein-coupled receptor signaling (RGS) domain that attenuates Gαs-coupled G protein-coupled receptor signaling. Here we delineate the structural organization of these RGS-PX proteins, revealing a protein family with a modular architecture that is conserved in all eukaryotes. The one exception to this is mammalian SNX19, which lacks the typical RGS structure but preserves all other domains. The PX domain is a sensor of membrane phosphoinositide lipids and we find that specific sequence alterations in the PX domains of the mammalian RGS-PX proteins, SNX13, SNX14, SNX19, and SNX25, confer differential phosphoinositide binding preferences. Although SNX13 and SNX19 PX domains bind the early endosomal lipid phosphatidylinositol 3-phosphate, SNX14 shows no membrane binding at all. Crystal structures of the SNX19 and SNX14 PX domains reveal key differences, with alterations in SNX14 leading to closure of the binding pocket to prevent phosphoinositide association. Our findings suggest a role for alternative membrane interactions in spatial control of RGS-PX proteins in cell signaling and trafficking.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Fosfatos de Fosfatidilinositol/química , Proteínas Recombinantes de Fusão/química , Nexinas de Classificação/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Movimento Celular , Sequência Conservada , Cristalografia por Raios X , Endossomos/química , Endossomos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fosfatos de Fosfatidilinositol/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismoRESUMO
TRAM/TICAM-2 is used by Toll-like receptor 4 (TLR4) as a bridging adaptor during the mammalian innate immune response. It recruits TRIF, another TIR domain-containing adaptor protein, to TLR4 via TIR domain interactions, which leads to the activation of transcription factors responsible for the production of type-1 interferon and cytokines. The molecular mechanisms of these dual interactions mediated by the TRAM TIR domain are not clear. To understand the molecular basis of TIR:TIR domain interactions, structural and biochemical studies of TRAM TIR domain are necessary, and require a functional soluble protein. In this paper, we report a successful purification and characterization of full-length TRAM. Because full-length TRAM likely contains unstructured regions that may be disadvantageous for structural studies, we also carried out a systematic construct design to determine the boundaries of the TRAM TIR domain. The truncated TRAM constructs were designed based on secondary structure predictions and screened by small-scale expression. Selected constructs were subjected to biophysical analyses. We show that the expressed TRAM TIR domain is functional using in vitro GST pull-down assays that demonstrate a physical interaction with the TLR4 TIR domain. We further show, by site-directed mutagenesis, that the "BB loop" regions of both the TRAM TIR domain and the TLR4 TIR domain are crucial for this physical interaction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bioquímica/métodos , Interferons/metabolismo , Proteínas Recombinantes/biossíntese , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Receptor 4 Toll-Like/químicaRESUMO
Erythroid Krüppel-like factor (EKLF) plays an important role in erythroid development by stimulating ß-globin gene expression. We have examined the details by which the minimal transactivation domain (TAD) of EKLF (EKLFTAD) interacts with several transcriptional regulatory factors. We report that EKLFTAD displays homology to the p53TAD and, like the p53TAD, can be divided into two functional subdomains (EKLFTAD1 and EKLFTAD2). Based on sequence analysis, we found that EKLFTAD2 is conserved in KLF2, KLF4, KLF5, and KLF15. In addition, we demonstrate that EKLFTAD2 binds the amino-terminal PH domain of the Tfb1/p62 subunit of TFIIH (Tfb1PH/p62PH) and four domains of CREB-binding protein/p300. The solution structure of the EKLFTAD2/Tfb1PH complex indicates that EKLFTAD2 binds Tfb1PH in an extended conformation, which is in contrast to the α-helical conformation seen for p53TAD2 in complex with Tfb1PH. These studies provide detailed mechanistic information into EKLFTAD functions as well as insights into potential interactions of the TADs of other KLF proteins. In addition, they suggest that not only have acidic TADs evolved so that they bind using different conformations on a common target, but that transitioning from a disordered to a more ordered state is not a requirement for their ability to bind multiple partners.
Assuntos
Fatores de Transcrição Kruppel-Like/química , Sequência de Aminoácidos , Sítios de Ligação , Calorimetria , Clonagem Molecular , Humanos , Células K562 , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismoRESUMO
The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work provides a solid basis for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers.
Assuntos
Domínio Catalítico , Íntrons , Splicing de RNA , Spliceossomos , Splicing de RNA/efeitos dos fármacos , Spliceossomos/metabolismo , Spliceossomos/efeitos dos fármacos , Humanos , Íntrons/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/químicaRESUMO
As for all non-segmented negative RNA viruses, rabies virus has its genome packaged in a linear assembly of nucleoprotein (N), named nucleocapsid. The formation of new nucleocapsids during virus replication in cells requires the production of soluble N protein in complex with its phosphoprotein (P) chaperone. In this study, we reconstituted a soluble heterodimeric complex between an armless N protein of rabies virus (RABV), lacking its N-terminal subdomain (NNT-ARM), and a peptide encompassing the N0 chaperon module of the P protein. We showed that the chaperone module undergoes a disordered-order transition when it assembles with N0 and measured an affinity in the low nanomolar range using a competition assay. We solved the crystal structure of the complex at a resolution of 2.3 Å, unveiling the details of the conserved interfaces. MD simulations showed that both the chaperon module of P and RNA-mediated polymerization reduced the ability of the RNA binding cavity to open and close. Finally, by reconstituting a complex with full-length P protein, we demonstrated that each P dimer could independently chaperon two N0 molecules.
Assuntos
Vírus da Raiva , Vírus da Raiva/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Proteínas do Nucleocapsídeo/genética , Chaperonas Moleculares/metabolismo , Fosfoproteínas/genética , RNA/metabolismo , RNA Viral/metabolismoRESUMO
The Apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that include Toxoplasma gondii, Plasmodium, and Cryptosporidium spp., which infect humans and animals and cause severe parasitic diseases. Available therapeutics against these diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. We use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index when used against T. gondii. We have identified TgPRP4K of T. gondii as the primary target of altiratinib using genetic target deconvolution, which highlighted key residues within the kinase catalytic site that conferred drug resistance when mutated. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for TgPRP4K and for its Plasmodium falciparum counterpart, PfCLK3. Our data identified structural features critical for binding of the other PfCLK3 inhibitor, TCMDC-135051. Consistent with the splicing control activity of this kinase family, we have shown that altiratinib can cause global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum. Thus, our data establish parasitic PRP4K/CLK3 as a potential pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.
Assuntos
Criptosporidiose , Cryptosporidium , Malária Falciparum , Toxoplasma , Inibidores da Angiogênese/uso terapêutico , Animais , Humanos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum , Inibidores de Proteínas Quinases/farmacologia , Spliceossomos , Toxoplasma/genéticaRESUMO
To understand the dynamic interactions between the phosphoprotein (P) and the nucleoprotein (N) within the transcription/replication complex of the Paramyxoviridae and to decipher their roles in regulating viral multiplication, we characterized the structural properties of the C-terminal X domain (PXD) of Nipah (NiV) and Hendra virus (HeV) P protein. In crystals, isolated NiV PXD adopted a two-helix dimeric conformation, which was incompetent for binding its partners, but in complex with the C-terminal intrinsically disordered tail of the N protein (NTAIL), it folded into a canonical 3H bundle conformation. In solution, SEC-MALLS, SAXS and NMR spectroscopy experiments indicated that both NiV and HeV PXD were larger in size than expected for compact proteins of the same molecular mass and were in conformational exchange between a compact three-helix (3H) bundle and partially unfolded conformations, where helix α3 is detached from the other two. Some measurements also provided strong evidence for dimerization of NiV PXD in solution but not for HeV PXD. Ensemble modeling of experimental SAXS data and statistical-dynamical modeling reconciled all these data, yielding a model where NiV and HeV PXD exchanged between different conformations, and where NiV but not HeV PXD formed dimers. Finally, recombinant NiV comprising a chimeric P carrying HeV PXD was rescued and compared with parental NiV. Experiments carried out in cellula demonstrated that the replacement of PXD did not significantly affect the replication dynamics while caused a slight virus attenuation, suggesting a possible role of the dimerization of NiV PXD in viral replication.