RESUMEN
An important aspect in the analytical characterization of protein therapeutics is the comprehensive characterization of higher order structure (HOS). Nuclear magnetic resonance (NMR) is arguably the most sensitive method for fingerprinting HOS of a protein in solution. Traditionally, (1)H-(15)N or (1)H-(13)C correlation spectra are used as a "structural fingerprint" of HOS. Here, we demonstrate that protein fingerprint by line shape enhancement (PROFILE), a 1D (1)H NMR spectroscopy fingerprinting approach, is superior to traditional two-dimensional methods using monoclonal antibody samples and a heavily glycosylated protein therapeutic (Epoetin Alfa). PROFILE generates a high resolution structural fingerprint of a therapeutic protein in a fraction of the time required for a 2D NMR experiment. The cross-correlation analysis of PROFILE spectra allows one to distinguish contributions from HOS vs protein heterogeneity, which is difficult to accomplish by 2D NMR. We demonstrate that the major analytical limitation of two-dimensional methods is poor selectivity, which renders these approaches problematic for the purpose of fingerprinting large biological macromolecules.
Asunto(s)
Técnicas de Química Analítica/normas , Espectroscopía de Resonancia Magnética/normas , Proteínas/química , Técnicas de Química Analítica/tendencias , Conformación ProteicaRESUMEN
BACKGROUND: Pneumococcus, meningococcus, and Haemophilus influenzae cause a similar spectrum of infections in the ear, lung, blood, and brain. They share cross-reactive antigens that bind to the laminin receptor of the blood-brain barrier as a molecular basis for neurotropism, and this step in pathogenesis was addressed in vaccine design. METHODS: Biologically active peptides derived from choline-binding protein A (CbpA) of pneumococcus were identified and then genetically fused to L460D pneumolysoid. The fusion construct was tested for vaccine efficacy in mouse models of nasopharyngeal carriage, otitis media, pneumonia, sepsis, and meningitis. RESULTS: The CbpA peptide-L460D pneumolysoid fusion protein was more broadly immunogenic than pneumolysoid alone, and antibodies were active in vitro against Streptococcus pneumoniae, Neisseria meningitidis, and H. influenzae. Passive and active immunization protected mice from pneumococcal carriage, otitis media, pneumonia, bacteremia, meningitis, and meningococcal sepsis. CONCLUSIONS: The CbpA peptide-L460D pneumolysoid fusion protein was broadly protective against pneumococcal infection, with the potential for additional protection against other meningeal pathogens.
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Proteínas Bacterianas/inmunología , Portador Sano/prevención & control , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/inmunología , Estreptolisinas/inmunología , Toxoides/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/genética , Protección Cruzada , Modelos Animales de Enfermedad , Femenino , Haemophilus influenzae/inmunología , Ratones , Ratones Endogámicos BALB C , Neisseria meningitidis/inmunología , Vacunas Neumococicas/administración & dosificación , Vacunas Neumococicas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Streptococcus pneumoniae/inmunología , Estreptolisinas/genética , Toxoides/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.
Asunto(s)
Anticuerpos Monoclonales/química , Espectroscopía de Resonancia Magnética/métodos , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
Restoration of p53 function through the disruption of the MDM2-p53 protein complex is a promising strategy for the treatment of various types of cancer. Here, we present kinetic, thermodynamic, and structural rationale for the remarkable potency of a new class of MDM2 inhibitors, the piperidinones. While these compounds bind to the same site as previously reported for small molecule inhibitors, such as the Nutlins, data presented here demonstrate that the piperidinones also engage the N-terminal region (residues 10-16) of human MDM2, in particular, Val14 and Thr16. This portion of MDM2 is unstructured in both the apo form of the protein and in MDM2 complexes with p53 or Nutlin, but adopts a novel ß-strand structure when complexed with the piperidinones. The ordering of the N-terminus upon binding of the piperidinones extends the current model of MDM2-p53 interaction and provides a new route to rational design of superior inhibitors.
Asunto(s)
Piperidinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Cristalografía por Rayos X , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Piperidinas/síntesis química , Piperidinas/química , Relación Estructura-Actividad , TermodinámicaRESUMEN
γ-Secretase modulators (GSMs) have received much attention as potential therapeutic agents for Alzheimer's disease (AD). GSMs increase the ratio between short and long forms of the amyloid-ß (Aß) polypeptides produced by γ-secretase and thereby decrease the amount of the toxic amyloid species. However, the mechanism of action of these agents is still poorly understood. One recent paper [Richter et al. (2010) Proc. Natl. Acad. Sci. U. S. A.107, 14597-14602] presented data that were interpreted to support direct binding of the GSM sulindac sulfide to Aß(42), supporting the notion that GSM action is linked to direct binding of these compounds to the Aß domain of its immediate precursor, the 99-residue C-terminal domain of the amyloid precursor protein (C99, also known as the ß-CTF). Here, contrasting results are presented that indicate there is no interaction between monomeric sulindac sulfide and monomeric forms of Aß42. Instead, it was observed that sulindac sulfide is itself prone to form aggregates that can bind nonspecifically to Aß42 and trigger its aggregation. This observation, combined with data from previous work [Beel et al. (2009) Biochemistry48, 11837-11839], suggests both that the poor behavior of some NSAID-based GSMs in solution may obscure results of binding assays and that NSAID-based GSMs do not function by directly targeting C99. It was also observed that another GSM, flurbiprofen, fails to bind to monomeric Aß42 or to C99 reconstituted into bilayered lipid vesicles. These results disfavor the hypothesis that these NSAID-based GSMs exert their modulatory effect by directly targeting a site located in the Aß42 domain of free C99.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Inhibidores Enzimáticos/farmacología , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Humanos , Cinética , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Evidence that certain gamma-secretase modulators (GSMs) target the 99-residue C-terminal domain (C99) of the amyloid precursor protein, a substrate of gamma-secretase, but not the protease complex itself has been presented [Kukar, T. L., et al. (2008) Nature 453, 925-929]. Here, NMR results demonstrate a lack of specific binding of these GSMs to monodisperse C99 in LMPG micelles. In addition, results indicate that C99 was likely to have been aggregated in some of the key experiments of the previous work and that binding of GSMs to these C99 aggregates is also of a nonspecific nature.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/química , Precursor de Proteína beta-Amiloide/química , Humanos , Micelas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Unión Proteica , Especificidad por SustratoRESUMEN
INTRODUCTION: Pharmacovigilance is essential to monitoring the safety profiles of authorized medicines. Compared with small-molecule drugs, biological drugs are more complex, more susceptible to structural variability due to manufacturing processes, and have the potential to induce immune-related reactions, underscoring the importance of safety monitoring for these products. Although highly similar to reference products, biosimilars are not expected to be structurally identical. For these reasons, proper reporting of potential adverse drug reactions (ADRs) using distinguishable names and batch numbers is essential for accurate tracing of all biological drugs. To address the need for robust pharmacovigilance, the European Parliament and Council of the European Union provided legislation regarding pharmacovigilance of biologics in 2010. AREAS COVERED: This narrative review examines the current state of pharmacovigilance for biologics in the European Union (EU) and discusses relevant information on pharmacovigilance of biosimilars, the current EU pharmacovigilance system, and areas that could be improved. EXPERT OPINION: Although steps have been taken to improve pharmacovigilance of biologics in the EU, several enhancements can still be made, including additional training for healthcare professionals on ADR reporting, the use of 2D barcodes that enhance traceability, and an open discussion of potentially missed opportunities in the pharmacovigilance of biosimilars.
Asunto(s)
Productos Biológicos/efectos adversos , Biosimilares Farmacéuticos/efectos adversos , Farmacovigilancia , Sistemas de Registro de Reacción Adversa a Medicamentos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Unión Europea , HumanosRESUMEN
The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.
Asunto(s)
Anticuerpos Monoclonales/química , Biofarmacia/normas , Laboratorios/normas , Espectroscopía de Resonancia Magnética/métodos , Humanos , Reproducibilidad de los ResultadosRESUMEN
Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.
Asunto(s)
Analgésicos/aislamiento & purificación , Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Péptidos/química , Bloqueadores de los Canales de Sodio/aislamiento & purificación , Venenos de Araña/química , Potenciales de Acción/efectos de los fármacos , Sustitución de Aminoácidos , Analgésicos/farmacología , Animales , Capsaicina/farmacología , Línea Celular , Evaluación Preclínica de Medicamentos , Ganglios Espinales/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Fibras Nerviosas Amielínicas/efectos de los fármacos , Resonancia Magnética Nuclear Biomolecular , Técnicas de Placa-Clamp , Estimulación Física , Ingeniería de Proteínas , Proteínas Recombinantes/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Relación Estructura-Actividad , Tetrodotoxina/farmacologíaRESUMEN
Fragment-based drug discovery (FBDD) has become a widely used tool in small-molecule drug discovery efforts. One of the most commonly used biophysical methods in detecting weak binding of fragments is nuclear magnetic resonance (NMR) spectroscopy. In particular, FBDD performed with (19)F NMR-based methods has been shown to provide several advantages over (1)H NMR using traditional magnetization-transfer and/or two-dimensional methods. Here, we demonstrate the utility and power of (19)F-based fragment screening by detailing the identification of a second-site fragment through (19)F NMR screening that binds to a specific pocket of the aspartic acid protease, ß-secretase (BACE-1). The identification of this second-site fragment allowed the undertaking of a fragment-linking approach, which ultimately yielded a molecule exhibiting a more than 360-fold increase in potency while maintaining reasonable ligand efficiency and gaining much improved selectivity over cathepsin-D (CatD). X-ray crystallographic studies of the molecules demonstrated that the linked fragments exhibited binding modes consistent with those predicted from the targeted screening approach, through-space NMR data, and molecular modeling.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Espectroscopía de Resonancia Magnética/métodos , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Flúor , Modelos Moleculares , Resonancia por Plasmón de SuperficieRESUMEN
The efficacy of therapeutic antibodies that induce antibody-dependent cellular cytotoxicity can be improved by reduced fucosylation. Consequently, fucosylation is a critical product attribute of monoclonal antibodies produced as protein therapeutics. Small molecule fucosylation inhibitors have also shown promise as potential therapeutics in animal models of tumors, arthritis, and sickle cell disease. Potent small molecule metabolic inhibitors of cellular protein fucosylation, 6,6,6-trifluorofucose per-O-acetate and 6,6,6-trifluorofucose (fucostatin I), were identified that reduces the fucosylation of recombinantly expressed antibodies in cell culture in a concentration-dependent fashion enabling the controlled modulation of protein fucosylation levels. 6,6,6-Trifluorofucose binds at an allosteric site of GDP-mannose 4,6-dehydratase (GMD) as revealed for the first time by the X-ray cocrystal structure of a bound allosteric GMD inhibitor. 6,6,6-Trifluorofucose was found to be incorporated in place of fucose at low levels (<1%) in the glycans of recombinantly expressed antibodies. A fucose-1-phosphonate analog, fucostatin II, was designed that inhibits fucosylation with no incorporation into antibody glycans, allowing the production of afucosylated antibodies in which the incorporation of non-native sugar is completely absent-a key advantage in the production of therapeutic antibodies, especially biosimilar antibodies. Inhibitor structure-activity relationships, identification of cellular and inhibitor metabolites in inhibitor-treated cells, fucose competition studies, and the production of recombinant antibodies with varying levels of fucosylation are described.
Asunto(s)
Fucosa/metabolismo , Hidroliasas/metabolismo , Bibliotecas de Moléculas Pequeñas , Animales , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Fucosa/antagonistas & inhibidores , Guanosina Difosfato Manosa/metabolismo , Espectrometría de Masas , Estructura Molecular , Resonancia por Plasmón de SuperficieAsunto(s)
Biosimilares Farmacéuticos/administración & dosificación , Aprobación de Drogas , Farmacovigilancia , Biosimilares Farmacéuticos/efectos adversos , Biosimilares Farmacéuticos/economía , Costos de los Medicamentos , Desarrollo de Medicamentos , Humanos , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure.
Asunto(s)
Carbohidratos/análisis , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Nucleótidos/análisis , Tampones (Química) , Fucosa/análogos & derivados , Fucosa/análisis , Hexosafosfatos/análisis , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Estructura Molecular , Azúcares de Nucleósido Difosfato/análisis , Solventes/química , Electricidad Estática , Fosfatos de Azúcar/análisisRESUMEN
Fragment based drug discovery (FBDD) is a widely used tool for discovering novel therapeutics. NMR is a powerful means for implementing FBDD, and several approaches have been proposed utilizing (1)H-(15)N heteronuclear single quantum coherence (HSQC) as well as one-dimensional (1)H and (19)F NMR to screen compound mixtures against a target of interest. While proton-based NMR methods of fragment screening (FBS) have been well documented and are widely used, the use of (19)F detection in FBS has been only recently introduced (Vulpetti et al. J. Am. Chem. Soc.2009, 131 (36), 12949-12959) with the aim of targeting "fluorophilic" sites in proteins. Here, we demonstrate a more general use of (19)F NMR-based fragment screening in several areas: as a key tool for rapid and sensitive detection of fragment hits, as a method for the rapid development of structure-activity relationship (SAR) on the hit-to-lead path using in-house libraries and/or commercially available compounds, and as a quick and efficient means of assessing target druggability.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/química , Bases de Datos Factuales , Diseño de Fármacos , Flúor , Relación Estructura-Actividad Cuantitativa , Aminoquinolinas/química , Espectroscopía de Resonancia Magnética , Resonancia por Plasmón de SuperficieRESUMEN
Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys(1)-Cys(8), Cys(3)-Cys(6), Cys(2)-Cys(4), and Cys(5)-Cys(7). NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Disulfuros/química , Pliegue de Proteína , Animales , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Disulfuros/metabolismo , Hepcidinas , Humanos , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína/fisiologíaRESUMEN
HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.
Asunto(s)
ADN/química , Hepacivirus/enzimología , Proteínas no Estructurales Virales/metabolismo , Adenosina Trifosfatasas/química , Catálisis , Cromatografía/métodos , Cromatografía en Gel , Reactivos de Enlaces Cruzados/farmacología , ADN Helicasas/química , Técnicas In Vitro , Cinética , Luz , Desnaturalización de Ácido Nucleico , Estructura Terciaria de Proteína , Dispersión de Radiación , Factores de TiempoRESUMEN
Recent advances in instrumentation and isotope labeling methodology allow proteins up to 100 kDa in size to be studied in detail using NMR spectroscopy. Using 2H/13C/15N enrichment and selective methyl protonation, we show that newly developed 13C direct detection methods can be used to rapidly yield proton and carbon resonance assignments for the methyl groups of Val, Leu, and Ile residues. We present a highly sensitive 13C-detected CH3-TOCSY experiment that, in combination with standard 1H-detected backbone experiments, allows the full assignment of side chain resonances in methyl-protonated residues. Selective methyl protonation, originally developed by Kay and co-workers (Rosen, M. K.; Gardner, K. H.; Willis, R. C.; Parris, W. E.; Pawson, T.; Kay, L. E. J. Mol. Biol. 1996, 263, 627-636; Gardner, K. G.; Kay, L. E. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357-406; Goto, N. K.; Kay, L. E. Curr. Opin. Struct. Biol. 2000, 10, 585-592), improves the nuclear relaxation behavior of larger proteins compared to their fully protonated counterparts, allows significant simplification of spectra, and facilitates NOE assignments. Here, we demonstrate the usefulness of the 13C-detected CH3-TOCSY experiment through studies of (i) a medium-sized protein (CbpA-R1; 14 kDa) with a repetitive primary sequence that yields highly degenerate NMR spectra, and (ii) a larger, bimolecular protein complex (p21-KID/Cdk2; 45 kDa) at low concentration in a high ionic strength solution. Through the analysis of NOEs involving amide and Ile, Leu, and Val methyl protons, we determined the global fold of CbpA-R1, a bacterial protein that mediates the pathogenic effects of Streptococcus pneumoniae, demonstrating that this approach can significantly reduce the time required to determine protein structures by NMR.
Asunto(s)
Carbono/química , Quinasa 2 Dependiente de la Ciclina/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Serina-Treonina Quinasas/química , Protones , Isótopos de Carbono , Entropía , Humanos , Conformación ProteicaRESUMEN
The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains. A crystal structure of the NS3 helicase domain (NS3h) was generated in the presence of a single-stranded oligonucleotide long enough to accommodate binding of two molecules of enzyme. Several amino acid residues at the interface of the two NS3h molecules were identified that appear to mediate a protein-protein interaction between domains 2 and 3 of adjacent molecules. Mutations were introduced into domain 3 to disrupt the putative interface and subsequently examined using an HCV subgenomic replicon, resulting in significant reduction in replication capacity. The mutations in domain 3 were then examined using recombinant NS3h in biochemical assays. The mutant enzyme showed RNA binding and RNA-stimulated ATPase activity that mirrored wild type NS3h. In DNA unwinding assays under single turnover conditions, the mutant NS3h exhibited a similar unwinding rate and only approximately 2-fold lower processivity than wild type NS3h. Overall biochemical activities of the mutant NS3h were similar to the wild type enzyme, which was not reflective of the large reduction in HCV replicative capacity observed in the biological experiment. Hence, the biological results suggest that the known biochemical properties associated with the helicase activity of NS3h do not reveal all of the likely biological roles of NS3 during HCV replication. Domain 3 of NS3 is implicated in protein-protein interactions that are necessary for HCV replication.