RESUMEN
The determination of a protein's structure is often a first step towards the development of a mechanistic understanding of its function. Considerable advances in computational protein structure prediction have been made in recent years, with AlphaFold2 (AF2) emerging as the primary tool used by researchers for this purpose. While AF2 generally predicts accurate structures of folded proteins, we present here a case where AF2 incorrectly predicts the structure of a small, folded and compact protein with high confidence. This protein, pro-interleukin-18 (pro-IL-18), is the precursor of the cytokine IL-18. Interestingly, the structure of pro-IL-18 predicted by AF2 matches that of the mature cytokine, and not the corresponding experimentally determined structure of the pro-form of the protein. Thus, while computational structure prediction holds immense promise for addressing problems in protein biophysics, there is still a need for experimental structure determination, even in the context of small well-folded, globular proteins.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Modelos Moleculares , Proteínas/química , Algoritmos , Interleucina-18/química , Programas InformáticosRESUMEN
Trastuzumab is a therapeutic monoclonal antibody developed to target human epidermal growth factor receptor 2 (HER2) present at higher levels in early cancers. Here we report the near complete resonance assignment of trastuzumab-scFab fragment backbone and the methyl groups of isoleucine, leucine and valine residues, as well as their stereo-assignments. The antibody fragment was produced using a single chain approach in Escherichia coli.
RESUMEN
Several interleukin-1 (IL-1) family members, including IL-1ß and IL-18, require processing by inflammasome-associated caspases to unleash their activities. Here, we unveil, by cryoelectron microscopy (cryo-EM), two major conformations of the complex between caspase-1 and pro-IL-18. One conformation is similar to the complex of caspase-4 and pro-IL-18, with interactions at both the active site and an exosite (closed conformation), and the other only contains interactions at the active site (open conformation). Thus, pro-IL-18 recruitment and processing by caspase-1 is less dependent on the exosite than the active site, unlike caspase-4. Structure determination by nuclear magnetic resonance uncovers a compact fold of apo pro-IL-18, which is similar to caspase-1-bound pro-IL-18 but distinct from cleaved IL-18. Binding sites for IL-18 receptor and IL-18 binding protein are only formed upon conformational changes after pro-IL-18 cleavage. These studies show how pro-IL-18 is selected as a caspase-1 substrate, and why cleavage is necessary for its inflammatory activity.
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Caspasa 1 , Microscopía por Crioelectrón , Interleucina-18 , Transducción de Señal , Interleucina-18/metabolismo , Caspasa 1/metabolismo , Humanos , Inflamasomas/metabolismo , Animales , Conformación Proteica , Unión Proteica , Sitios de Unión , Ratones , Receptores de Interleucina-18/metabolismo , Modelos Moleculares , Péptidos y Proteínas de Señalización IntercelularRESUMEN
While protein activity is traditionally studied with a major focus on the active site, the activity of enzymes has been hypothesized to be linked to the flexibility of adjacent regions, warranting more exploration into how the dynamics in these regions affects catalytic turnover. One such enzyme is Xylanase A (XylA), which cleaves hemicellulose xylan polymers by hydrolysis at internal ß-1,4-xylosidic linkages. It contains a "thumb" region whose flexibility has been suggested to affect the activity. The double mutation D11F/R122D was previously found to affect activity and potentially bias the thumb region to a more open conformation. We find that the D11F/R122D double mutation shows substrate-dependent effects, increasing activity on the non-native substrate ONPX2 but decreasing activity on its native xylan substrate. To characterize how the double mutant causes these kinetics changes, nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations were used to probe structural and flexibility changes. NMR chemical shift perturbations revealed structural changes in the double mutant relative to the wild-type, specifically in the thumb and fingers regions. Increased slow-timescale dynamics in the fingers region was observed as intermediate-exchange line broadening. Lipari-Szabo order parameters show negligible changes in flexibility in the thumb region in the presence of the double mutation. To help understand if there is increased energetic accessibility to the open state upon mutation, alchemical free energy simulations were employed that indicated thumb opening is more favorable in the double mutant. These studies aid in further characterizing how flexibility in adjacent regions affects the function of XylA.
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Endo-1,4-beta Xilanasas , Simulación de Dinámica Molecular , Mutación , Xilanos , Especificidad por Sustrato/genética , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/metabolismo , Mutación/genética , Xilanos/metabolismo , Xilanos/química , Dominio Catalítico/genética , Cinética , Conformación Proteica , Espectroscopía de Resonancia MagnéticaRESUMEN
Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.
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Apoptosomas , Caspasas , Caspasa 9/metabolismo , Apoptosomas/química , Caspasas/metabolismo , Apoptosis , Espectroscopía de Resonancia Magnética , Caspasa 3/metabolismoRESUMEN
InhA, the Mycobacterium tuberculosis enoyl-ACP reductase, is a target for the tuberculosis (TB) drug isoniazid (INH). InhA inhibitors that do not require KatG activation avoid the most common mechanism of INH resistance, and there are continuing efforts to fully elucidate the enzyme mechanism to drive inhibitor discovery. InhA is a member of the short-chain dehydrogenase/reductase superfamily characterized by a conserved active site Tyr, Y158 in InhA. To explore the role of Y158 in the InhA mechanism, this residue has been replaced by fluoroTyr residues that increase the acidity of Y158 up to â¼3200-fold. Replacement of Y158 with 3-fluoroTyr (3-FY) and 3,5-difluoroTyr (3,5-F2Y) has no effect on kcatapp/KMapp nor on the binding of inhibitors to the open form of the enzyme (Kiapp), whereas both kcatapp/KMapp and Kiapp are altered by seven-fold for the 2,3,5-trifluoroTyr variant (2,3,5-F3Y158 InhA). 19F NMR spectroscopy suggests that 2,3,5-F3Y158 is ionized at neutral pH indicating that neither the acidity nor ionization state of residue 158 has a major impact on catalysis or on the binding of substrate-like inhibitors. In contrast, Ki*app is decreased 6- and 35-fold for the binding of the slow-onset inhibitor PT504 to 3,5-F2Y158 and 2,3,5-F3Y158 InhA, respectively, indicating that Y158 stabilizes the closed form of the enzyme adopted by EI*. The residence time of PT504 is reduced â¼four-fold for 2,3,5-F3Y158 InhA compared to wild-type, and thus, the hydrogen bonding interaction of the inhibitor with Y158 is an important factor in the design of InhA inhibitors with increased residence times on the enzyme.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Antituberculosos/farmacología , Antituberculosos/química , Isoniazida/química , Isoniazida/farmacología , Dominio Catalítico , Proteínas Bacterianas/químicaRESUMEN
The HMQC pulse sequence and variants thereof have been exploited in studies of high molecular weight protein complexes, taking advantage of the fact that fast and slow relaxing magnetization components are sequestered along two distinct magnetization transfer pathways. Despite the simplicity of the HMQC scheme an even shorter version can be designed, based on elimination of the terminal refocusing period, as a further means of increasing signal. Here we present such an experiment, and show that significant sensitivity gains, in some cases by factors of two or more, are realized in studies of proteins varying in molecular masses from 100 kDa to 1 MDa.
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Proteínas , Isótopos de Carbono , Peso Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
Using deuterium-labeled stereochemical probes, we show that primary alkyltrifluoroborate nucleophiles undergo transmetalation to palladium exclusively via a stereoretentive pathway and that the resulting stereospecificity is broadly independent of electronic and steric effects. This stands in stark contrast to the stereochemical course of transmetalation for secondary alkyltrifluoroborates, which varies between net stereoretention and net stereoinversion depending upon the electronic properties of the supporting phosphine ligand, the electronic properties of the aryl electrophile, and the steric properties of the alkylboron nucleophile. In this study, we additionally show that the stereochemical course of transmetalation for secondary alkylboron reagents can be under reagent steric control, while no such steric control exists for analogous primary alkylboron nucleophiles. The combined study reveals fundamental mechanistic differences between transmetalations of primary and secondary alkylboron reagents in Pd-catalyzed Suzuki reactions.
RESUMEN
Elastin fibers assemble in the extracellular matrix from the precursor protein tropoelastin and provide the flexibility and spontaneous recoil required for arterial function. Unlike many proteins, a structure-function mechanism for elastin has been elusive. We have performed detailed NMR relaxation studies of the dynamics of the minielastins 24x' and 20x' using solution NMR, and of purified bovine elastin fibers in the presence and absence of mechanical stress using solid state NMR. The low sequence complexity of the minielastins enables us to determine average dynamical timescales and degrees of local ordering in the cross-link and hydrophobic modules separately using NMR relaxation by taking advantage of their residue-specific resolution. We find an extremely high degree of disorder, with order parameters for the entirety of the hydrophobic domains near zero, resembling that of simple chemical polymers and less than the order parameters that have been observed in other intrinsically disordered proteins. We find that average backbone order parameters in natural, purified elastin fibers are comparable to those found in 24x' and 20x' in solution. The difference in dynamics, compared with the minielastins, is that backbone correlation times are significantly slowed in purified elastin. Moreover, when elastin is mechanically stretched, the high chain disorder in purified elastin is retained, showing that any change in local ordering is below that detectable in our experiment. Combined with our previous finding of a 10-fold increase in the ordering of water when fully hydrated elastin fibers are stretched by 50%, these results support the hypothesis that stretch induced solvent ordering, i.e., the hydrophobic effect, is a key player in the elastic recoil of elastin as opposed to configurational entropy loss.
Asunto(s)
Tejido Elástico , Elastina , Animales , Bovinos , Matriz Extracelular , Interacciones Hidrofóbicas e Hidrofílicas , TropoelastinaRESUMEN
Proteins often interconvert between different conformations in ways critical to their function. Although manipulating such equilibria for biophysical study is often challenging, the application of pressure is a potential route to achieve such control by favoring the population of lower volume states. Here, we use this feature to study the interconversion of ARNT PAS-B Y456T, which undergoes a dramatic +3 slip in the ß-strand register as it switches between two stably folded conformations. Using high-pressure biomolecular NMR approaches, we obtained the first, to our knowledge, quantitative data testing two key hypotheses of this process: the slipped conformation is both smaller and less compressible than the wild-type equivalent, and the interconversion proceeds through a chiefly unfolded intermediate state. Data collected in steady-state pressure and time-resolved pressure-jump modes, including observed pressure-dependent changes in the populations of the two conformers and increased rate of interconversion between conformers, support both hypotheses. Our work exemplifies how these approaches, which can be generally applied to protein conformational switches, can provide unique information that is not easily accessible through other techniques.
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Pliegue de Proteína , Proteínas , Espectroscopía de Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular , Conformación ProteicaRESUMEN
Melanin and related polyphenolic pigments are versatile functional polymers that serve diverse aesthetic and protective roles across the living world. These polymeric pigments continue to inspire the development of adhesive, photonic, electronic and radiation-protective materials and coatings. The properties of these structures are dictated by covalent and non-covalent interactions in ways that, despite progress, are not fully understood. It remains a major challenge to direct oxidative polymerization of their precursors (amino acids, (poly-)phenols, thiols) toward specific structures. By taking advantage of supramolecular pre-organization of tyrosine-tripeptides and reactive sequestering of selected amino acids during enzymatic oxidation, we demonstrate the spontaneous formation of distinct new chromophores with optical properties that are far beyond the range of those found in biological melanins, in terms of color, UV absorbance and fluorescent emission.
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Colorantes Fluorescentes/química , Melaninas/química , Péptidos/química , Polifenoles/química , Secuencia de Aminoácidos , Aminoácidos/química , Microesferas , Oxidación-Reducción , Polimerizacion , Compuestos de Sulfhidrilo/química , Propiedades de SuperficieRESUMEN
Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light-oxygen-voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output domain. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 µs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in a loss of the interaction between the side chain of N414 and the backbone CâO of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.
Asunto(s)
Avena/química , Glutamina/química , Fototropinas/metabolismo , Desplegamiento Proteico/efectos de la radiación , Mononucleótido de Flavina/metabolismo , Enlace de Hidrógeno , Luz , Proteínas de la Membrana/metabolismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , Optogenética , Fototropinas/genética , Fototropinas/efectos de la radiación , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios Proteicos , Multimerización de Proteína/efectos de la radiaciónRESUMEN
Because of their potential as novel antibiotic agents, antimicrobial peptides (AMPs) have generated considerable interest. The mechanism of bacterial toxicity of AMPs often involves the disruption and/or permeabilization of the bacterial membrane; even those that act intracellularly first have to traverse the membrane. In this work we have explored the incorporation of the fluorinated aromatic amino acids fluoro-Phe and fluoro-Tyr into the Trp- and Arg-rich AMP tritrpticin, and investigated their role in the membrane binding properties and the antimicrobial activity of the peptide. Fluorinated peptides were obtained with good yield by recombinant expression of tritrpticin as a calmodulin-fusion protein in Escherichia coli. Cells were grown in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, and the peptides were released by proteolysis from the purified fusion protein. By using SDS micelles, as a simplified model of the bacterial cytoplasmic membrane, we could study the peptide-membrane interactions and the preferred location of individual fluorinated residues in the micelles by 19F NMR spectroscopy. Solvent-perturbation 19F NMR measurements revealed that para-fluoro-Phe residues are embedded deeply in the hydrophobic region of the micelles. On the other hand, 3-fluoro-Tyr residues introduced in tritrpticin were located near the surface of the micelles with high solvent exposure, while 2-fluoro-Tyr sidechains were less solvent exposed. In combination with the outcome of determinations of their antimicrobial activity, our 19F NMR results indicate that the higher solvent exposure of Tyr residues correlates with a decrease of the antimicrobial potency. This different role of Tyr can likely be extended from tritrpticin to other cationic AMPs.
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Péptidos Catiónicos Antimicrobianos/química , Flúor , Espectroscopía de Resonancia Magnética/métodos , Oligopéptidos/química , Tirosina/fisiología , Péptidos Catiónicos Antimicrobianos/toxicidad , Membrana Celular/metabolismo , Micelas , Oligopéptidos/metabolismo , Dodecil Sulfato de SodioRESUMEN
Transcription factors comprise a major reservoir of conformational disorder in the eukaryotic proteome. The hematopoietic master regulator PU.1 presents a well-defined model of the most common configuration of intrinsically disordered regions (IDRs) in transcription factors. We report that the structured DNA binding domain (DBD) of PU.1 regulates gene expression via antagonistic dimeric states that are reciprocally controlled by cognate DNA on the one hand and by its proximal anionic IDR on the other. The two conformers are mediated by distinct regions of the DBD without structured contributions from the tethered IDRs. Unlike DNA-bound complexes, the unbound dimer is markedly destabilized. Dimerization without DNA is promoted by progressive phosphomimetic substitutions of IDR residues that are phosphorylated in immune activation and stimulated by anionic crowding agents. These results suggest a previously unidentified, nonstructural role for charged IDRs in conformational control by mitigating electrostatic penalties that would mask the interactions of highly cationic DBDs.
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Proteínas Intrínsecamente Desordenadas/metabolismo , Multimerización de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , ADN/metabolismo , Retroalimentación Fisiológica , Humanos , Proteínas Intrínsecamente Desordenadas/química , Mutación/genética , Conformación Proteica , Dominios Proteicos , Estabilidad Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Espectroscopía de Protones por Resonancia Magnética , Electricidad Estática , Transactivadores/química , Transactivadores/genética , Activación TranscripcionalRESUMEN
Antimicrobial proteins such as S100A12 and S100A8/A9 are highly expressed and secreted by neutrophils during infection and participate in human immune response by sequestering transition metals. At neutral pH, S100A12 sequesters Zn2+ with nanomolar affinity, which is further enhanced upon calcium binding. We investigated the pH dependence of human S100A12 zinc sequestration by using Co2+ as a surrogate. Apo-S100A12 exhibits strong Co2+ binding between pHâ 7.0 and 10.0 that progressively diminishes as the pH is decreased to 5.3. Ca2+ -S100A12 can retain nanomolar Co2+ binding up to pHâ 5.7. NMR spectroscopic measurements revealed that calcium binding does not alter the side-chain protonation of the Co2+ /Zn2+ binding histidine residues. Instead, the calcium-mediated modulation is achieved by restraining pH-dependent conformational changes to EF loopâ 1, which contains Co2+ /Zn2+ binding Asp25. This calcium-induced enhancement of Co2+ /Zn2+ binding might assist in the promotion of antimicrobial activities in humans by S100 proteins during neutrophil activation under subneutral pH conditions.
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Calcio/farmacología , Cobalto/metabolismo , Proteína S100A12/química , Proteína S100A12/metabolismo , Zinc/metabolismo , Cobalto/química , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Relación Estructura-Actividad , Zinc/químicaRESUMEN
Influenza viruses cause a highly contagious respiratory disease in humans. The NS1 proteins of influenza A and B viruses (NS1A and NS1B proteins, respectively) are composed of two domains, a dimeric N-terminal domain and a C-terminal domain, connected by a flexible polypeptide linker. Here we report the 2.0-Å X-ray crystal structure and nuclear magnetic resonance studies of the NS1B C-terminal domain, which reveal a novel and unexpected basic RNA-binding site that is not present in the NS1A protein. We demonstrate that single-site alanine replacements of basic residues in this site lead to reduced RNA-binding activity, and that recombinant influenza B viruses expressing these mutant NS1B proteins are severely attenuated in replication. This novel RNA-binding site of NS1B is required for optimal influenza B virus replication. Most importantly, this study reveals an unexpected RNA-binding function in the C-terminal domain of NS1B, a novel function that distinguishes influenza B viruses from influenza A viruses.
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Virus de la Influenza A/genética , Virus de la Influenza B/genética , Mutación , ARN Viral/química , Proteínas de Unión al ARN/química , Proteínas no Estructurales Virales/química , Células A549 , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Expresión Génica , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza B/metabolismo , Cinética , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Especificidad por Sustrato , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
The increase in antibiotic-resistant bacterial infections has prompted significant academic research into new therapeutic agents targeted against these pathogens. Antimicrobial peptides (AMPs) appear as promising candidates, due their potent antimicrobial activity and their ubiquitous presence in almost all organisms. Tritrpticin is a member of this family of peptides and has been shown to exert a strong antimicrobial activity against several bacterial strains. Tritrpticin's main structural characteristic is the presence of three consecutive Trp residues at the center of the peptide. These residues play an important role in the activity of tritrpticin against Escherichia coli. In this work, a recombinant version of tritrpticin was produced in E. coli using calmodulin as a fusion protein expression tag to overcome the toxicity of the peptide. When used in combination with glyphosate, an inhibitor of the endogenous synthesis of aromatic amino acids, this expression system allowed for the incorporation of fluorinated Trp analogs at very high levels (>90%). The antimicrobial activity of the 4-, 5- and 6-fluoro-Trp-containing tritrpticins against E. coli was as strong as the activity of the native peptide. Similarly, the tritrpticin analogs exhibited comparable abilities to perturb and permeabilize synthetic lipid bilayers as well as the outer and inner membrane of E. coli. Furthermore, the use of 19F NMR spectroscopy established that each individual fluoro-Trp residue interacts differently with SDS micelles, supporting the idea that each Trp in the original tritrpticin plays a different role in the perturbing/permeabilizing activity of the peptide. Moreover, our work demonstrates that the use of fluoro-Trp in solvent perturbation 19F NMR experiments provides detailed site-specific information on the insertion of the Trp residues in biological membrane mimetics. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.
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Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Oligopéptidos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Antibacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Calmodulina/genética , Calmodulina/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Glicina/análogos & derivados , Glicina/farmacología , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Oligopéptidos/biosíntesis , Oligopéptidos/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Triptófano/análogos & derivados , Triptófano/metabolismo , GlifosatoRESUMEN
We have developed an online NMR / X-ray Structure Pair Data Repository. The NIGMS Protein Structure Initiative (PSI) has provided many valuable reagents, 3D structures, and technologies for structural biology. The Northeast Structural Genomics Consortium was one of several PSI centers. NESG used both X-ray crystallography and NMR spectroscopy for protein structure determination. A key goal of the PSI was to provide experimental structures for at least one representative of each of hundreds of targeted protein domain families. In some cases, structures for identical (or nearly identical) constructs were determined by both NMR and X-ray crystallography. NMR spectroscopy and X-ray diffraction data for 41 of these "NMR / X-ray" structure pairs determined using conventional triple-resonance NMR methods with extensive sidechain resonance assignments have been organized in an online NMR / X-ray Structure Pair Data Repository. In addition, several NMR data sets for perdeuterated, methyl-protonated protein samples are included in this repository. As an example of the utility of this repository, these data were used to revisit questions about the precision and accuracy of protein NMR structures first outlined by Levy and coworkers several years ago (Andrec et al., Proteins 2007;69:449-465). These results demonstrate that the agreement between NMR and X-ray crystal structures is improved using modern methods of protein NMR spectroscopy. The NMR / X-ray Structure Pair Data Repository will provide a valuable resource for new computational NMR methods development.
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Cristalografía por Rayos X , Bases de Datos de Proteínas , Resonancia Magnética Nuclear Biomolecular , Modelos Moleculares , Conformación Proteica , Proteínas/químicaRESUMEN
RAS binding is a critical step in the activation of BRAF protein serine/threonine kinase and stimulation of the mitogen-activated protein kinase signaling pathway. Mutations in both RAS and BRAF are associated with many human cancers. Here, we report the solution nuclear magnetic resonance (NMR) and X-ray crystal structures of the RAS-binding domain (RBD) from human BRAF. We further studied the complex between BRAF RBD and the GppNHp bound form of HRAS in solution. Backbone, side-chain, and (19)F NMR chemical shift perturbations reveal unexpected changes distal to the RAS-binding face that extend through the core of the RBD structure. Moreover, backbone amide hydrogen/deuterium exchange NMR data demonstrate conformational ensemble changes in the RBD core structure upon complex formation. These changes in BRAF RBD reveal a basis for allosteric regulation of BRAF structure and function, and suggest a mechanism by which RAS binding can signal the drastic domain rearrangements required for activation of BRAF kinase.
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Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas p21(ras)/química , Regulación Alostérica , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Transducción de SeñalRESUMEN
We report alterations to the murine leukemia virus (MLV) integrase (IN) protein that successfully result in decreasing its integration frequency at transcription start sites and CpG islands, thereby reducing the potential for insertional activation. The host bromo and extraterminal (BET) proteins Brd2, 3 and 4 interact with the MLV IN protein primarily through the BET protein ET domain. Using solution NMR, protein interaction studies, and next generation sequencing, we show that the C-terminal tail peptide region of MLV IN is important for the interaction with BET proteins and that disruption of this interaction through truncation mutations affects the global targeting profile of MLV vectors. The use of the unstructured tails of gammaretroviral INs to direct association with complexes at active promoters parallels that used by histones and RNA polymerase II. Viruses bearing MLV IN C-terminal truncations can provide new avenues to improve the safety profile of gammaretroviral vectors for human gene therapy.