RESUMEN
Intrinsically disordered regions of proteins often mediate important protein-protein interactions. However, the folding-upon-binding nature of many polypeptide-protein interactions limits the ability of modeling tools to predict the three-dimensional structures of such complexes. To address this problem, we have taken a tandem approach combining NMR chemical shift data and molecular simulations to determine the structures of peptide-protein complexes. Here, we use the MELD (Modeling Employing Limited Data) technique applied to polypeptide complexes formed with the extraterminal domain (ET) of bromo and extraterminal domain (BET) proteins, which exhibit a high degree of binding plasticity. This system is particularly challenging as the binding process includes allosteric changes across the ET receptor upon binding, and the polypeptide binding partners can adopt different conformations (e.g., helices and hairpins) in the complex. In a blind study, the new approach successfully modeled bound-state conformations and binding poses, using only protein receptor backbone chemical shift data, in excellent agreement with experimentally determined structures for moderately tight (Kd â¼100 nM) binders. The hybrid MELD + NMR approach required additional peptide ligand chemical shift data for weaker (Kd â¼250 µM) peptide binding partners. AlphaFold also successfully predicts the structures of some of these peptide-protein complexes. However, whereas AlphaFold can provide qualitative peptide rankings, MELD can directly estimate relative binding affinities. The hybrid MELD + NMR approach offers a powerful new tool for structural analysis of protein-polypeptide complexes involving disorder-to-order transitions upon complex formation, which are not successfully modeled with most other complex prediction methods, providing both the 3D structures of peptide-protein complexes and their relative binding affinities.
Asunto(s)
Simulación de Dinámica Molecular , Péptidos , Unión Proteica , Proteínas/química , Estructura Secundaria de Proteína , Conformación ProteicaRESUMEN
NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Proteínas de la Membrana , Modelos Moleculares , Conformación Proteica , Programas Informáticos , Biología Computacional , Aprendizaje Automático , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Pliegue de Proteína , Análisis de Secuencia de ProteínaRESUMEN
As complications associated with antibiotic resistance have intensified, copper (Cu) is attracting attention as an antimicrobial agent. Recent studies have shown that copper surfaces decrease microbial burden, and host macrophages use Cu to increase bacterial killing. Not surprisingly, microbes have evolved mechanisms to tightly control intracellular Cu pools and protect against Cu toxicity. Here, we identified two genes (copB and copL) encoded within the Staphylococcus aureus arginine-catabolic mobile element (ACME) that we hypothesized function in Cu homeostasis. Supporting this hypothesis, mutational inactivation of copB or copL increased copper sensitivity. We found that copBL are co-transcribed and that their transcription is increased during copper stress and in a strain in which csoR, encoding a Cu-responsive transcriptional repressor, was mutated. Moreover, copB displayed genetic synergy with copA, suggesting that CopB functions in Cu export. We further observed that CopL functions independently of CopB or CopA in Cu toxicity protection and that CopL from the S. aureus clone USA300 is a membrane-bound and surface-exposed lipoprotein that binds up to four Cu+ ions. Solution NMR structures of the homologous Bacillus subtilis CopL, together with phylogenetic analysis and chemical-shift perturbation experiments, identified conserved residues potentially involved in Cu+ coordination. The solution NMR structure also revealed a novel Cu-binding architecture. Of note, a CopL variant with defective Cu+ binding did not protect against Cu toxicity in vivo Taken together, these findings indicate that the ACME-encoded CopB and CopL proteins are additional factors utilized by the highly successful S. aureus USA300 clone to suppress copper toxicity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Cobre/toxicidad , Operón/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Membrana Celular/efectos de los fármacos , Cobre/metabolismo , Staphylococcus aureus/metabolismoRESUMEN
Members of an important group of industrial enzymes, Rhizopus lipases, exhibit valuable hydrolytic features that underlie their biological functions. Particularly important is their N-terminal polypeptide segment (NTPS), which is required for secretion and proper folding but is removed in the process of enzyme maturation. A second common feature of this class of lipases is the α-helical "lid", which regulates the accessibility of the substrate to the enzyme active site. Some Rhizopus lipases also exhibit "interfacial activation" by micelle and/or aggregate surfaces. While it has long been recognized that the NTPS is critical for function, its dynamic features have frustrated efforts to characterize its structure by X-ray crystallography. Here, we combine nuclear magnetic resonance spectroscopy and X-ray crystallography to determine the structure and dynamics of Rhizopus chinensis lipase (RCL) with its 27-residue NTPS prosequence (r27RCL). Both r27RCL and the truncated mature form of RCL (mRCL) exhibit biphasic interfacial activation kinetics with p-nitrophenyl butyrate (pNPB). r27RCL exhibits a substrate binding affinity significantly lower than that of mRCL due to stabilization of the closed lid conformation by the NTPS. In contrast to previous predictions, the NTPS does not enhance lipase activity by increasing surface hydrophobicity but rather inhibits activity by forming conserved interactions with both the closed lid and the core protein structure. Single-site mutations and kinetic studies were used to confirm that the NTPS serves as internal competitive inhibitor and to develop a model of the associated process of interfacial activation. These structure-function studies provide the basis for engineering RCL lipases with enhanced catalytic activities.
Asunto(s)
Proteínas Fúngicas/química , Microbiología Industrial , Lipasa/química , Péptidos/química , Rhizopus/enzimología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidrólisis , Cinética , Lipasa/genética , Lipasa/metabolismo , Resonancia Magnética Nuclear Biomolecular , Péptidos/genética , Péptidos/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
CASP13 has investigated the impact of sparse NMR data on the accuracy of protein structure prediction. NOESY and 15 N-1 H residual dipolar coupling data, typical of that obtained for 15 N,13 C-enriched, perdeuterated proteins up to about 40 kDa, were simulated for 11 CASP13 targets ranging in size from 80 to 326 residues. For several targets, two prediction groups generated models that are more accurate than those produced using baseline methods. Real NMR data collected for a de novo designed protein were also provided to predictors, including one data set in which only backbone resonance assignments were available. Some NMR-assisted prediction groups also did very well with these data. CASP13 also assessed whether incorporation of sparse NMR data improves the accuracy of protein structure prediction relative to nonassisted regular methods. In most cases, incorporation of sparse, noisy NMR data results in models with higher accuracy. The best NMR-assisted models were also compared with the best regular predictions of any CASP13 group for the same target. For six of 13 targets, the most accurate model provided by any NMR-assisted prediction group was more accurate than the most accurate model provided by any regular prediction group; however, for the remaining seven targets, one or more regular prediction method provided a more accurate model than even the best NMR-assisted model. These results suggest a novel approach for protein structure determination, in which advanced prediction methods are first used to generate structural models, and sparse NMR data is then used to validate and/or refine these models.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Proteínas/química , Algoritmos , Simulación por Computador , Cristalografía por Rayos X , Reproducibilidad de los ResultadosRESUMEN
Deficits in DNA damage-repair pathways are the root cause of several human cancers. In mammalian cells, DNA double-strand break repair is carried out by multiple mechanisms, including homologous recombination (HR). The partner and localizer of BRCA2 (PALB2), which is an essential factor for HR, binds to the breast cancer susceptibility 1 (BRCA1) protein at DNA double-strand breaks. At the break site, PALB2 also associates with the breast cancer susceptibility 2 (BRCA2) protein to form a multiprotein complex that facilitates HR. The BRCA1-PALB2 interaction is mediated by association of predicted helical coiled-coil regions in both proteins. PALB2 can also homodimerize through the formation of a coiled coil by the self-association of helical elements at the N-terminus of the PALB2 protein, and this homodimerization has been proposed to regulate the efficiency of HR. We have produced a segment of PALB2, designated PALB2cc (PALB2 coiled coil segment) that forms α-helical structures, which assemble into stable homodimers. PALB2cc also forms heterodimers with a helical segment of BRCA1, called BRCA1cc (BRCA1 coiled coil segment). The three-dimensional structure of the homodimer formed by PALB2cc was determined by solution NMR spectroscopy. This PALB2cc homodimer is a classical antiparallel coiled-coil leucine zipper. NMR chemical-shift perturbation studies were used to study dimer formation for both the PALB2cc homodimer and the PALB2cc/BRCA1cc heterodimer. The mutation of residue Leu24 of PALB2cc significantly reduces its homodimer stability, but has a more modest effect on the stability of the heterodimer formed between PALB2cc and BRCA1cc. We show that mutation of Leu24 leads to genomic instability and reduced cell viability after treatment with agents that induce DNA double-strand breaks. These studies may allow the identification of distinct mutations of PALB2cc that selectively disrupt homodimeric versus heterodimeric interactions, and reveal the specific role of PALB2cc homodimerization in HR.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi/química , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Multimerización de Proteína , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Animales , Linfocitos B/metabolismo , Proteína BRCA1 , Células Cultivadas , Cristalografía por Rayos X , Ratones , Conformación ProteicaRESUMEN
Ambidoxin is a designed, minimal dodecapeptide consisting of alternating L and D amino acids that binds a 4Fe-4S cluster through ligand-metal interactions and an extensive network of second-shell hydrogen bonds. The peptide can withstand hundreds of oxidation-reduction cycles at room temperature. Ambidoxin suggests how simple, prebiotic peptides may have achieved robust redox catalysis on the early Earth.
Asunto(s)
Proteínas Hierro-Azufre/química , Técnicas Electroquímicas , Transporte de Electrón , Ligandos , Oxidación-ReducciónRESUMEN
The nature of flexibility in the helix-turn-helix region of E. coli trp aporepressor has been unexplained for many years. The original ensemble of nuclear magnetic resonance (NMR structures showed apparent disorder, but chemical shift and relaxation measurements indicated a helical region. Nuclear Overhauser effect (NOE) data for a temperature-sensitive mutant showed more helical character in its helix-turn-helix region, but nevertheless also led to an apparently disordered ensemble. However, conventional NMR structure determination methods require all structures in the ensemble to be consistent with every NOE simultaneously. This work uses an alternative approach in which some structures of the ensemble are allowed to violate some NOEs to permit modeling of multiple conformational states that are in dynamic equilibrium. Newly measured NOE data for wild-type aporepressor are used as time-averaged distance restraints in molecular dynamics simulations to generate an ensemble of helical conformations that is more consistent with the observed NMR data than the apparent disorder in the previously reported NMR structures. The results indicate the presence of alternating helical conformations that provide a better explanation for the flexibility of the helix-turn-helix region of trp aporepressor. Structures representing these conformations have been deposited with PDB ID: 5TM0. Proteins 2017; 85:731-740. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/química , Proteínas Represoras/química , Triptófano/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Escherichia coli/metabolismo , Cinética , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/metabolismo , Triptófano/metabolismoRESUMEN
BACKGROUND: In order to use most modern methods of NMR spectroscopy to study protein structure and dynamics, isotope-enriched protein samples are essential. Especially for larger proteins (>20 kDa), perdeuterated and Ile (δ1), Leu, and Val methyl-protonated protein samples are required for suppressing nuclear relaxation to provide improved spectral quality, allowing key backbone and side chain resonance assignments needed for protein structure and dynamics studies. Escherichia coli and Pichia pastoris are two of the most popular expression systems for producing isotope-enriched, recombinant protein samples for NMR investigations. The P. pastoris system can be used to produce (13)C, (15)N-enriched and even (2)H,(13)C, (15)N-enriched protein samples, but efficient methods for producing perdeuterated proteins with Ile (δ1), Leu and Val methyl-protonated groups in P. pastoris are still unavailable. Glycosylation heterogeneity also provides challenges to NMR studies. E. coli expression systems are efficient for overexpressing perdeuterated and Ile (δ1), Leu, Val methyl-protonated protein samples, but are generally not successful for producing secreted eukaryotic proteins with native disulfide bonds. RESULTS: The 33 kDa protein-Rhizopus chinensis lipase (RCL), an important industrial enzyme, was produced using both P. pastoris and E. coli BL21 trxB (DE3) systems. Samples produced from both systems exhibit identical native disulfide bond formation and similar 2D NMR spectra, indicating similar native protein folding. The yield of (13)C, (15)N-enriched r27RCL produced using P. pastoris was 1.7 times higher that obtained using E. coli, while the isotope-labeling efficiency was ~15 % lower. Protein samples produced in P. pastoris exhibit O-glycosylation, while the protein samples produced in E. coli were not glycosylated. The specific activity of r27RCL from P. pastoris was ~1.4 times higher than that produced in E. coli. CONCLUSIONS: These data demonstrate efficient production of (2)H, (13)C, (15)N-enriched, Ile (δ1), Leu, Val methyl-protonated eukaryotic protein r27RCL with native disulfides using the E. coli BL21 trxB (DE3) system. For certain NMR studies, particularly efforts for resonance assignments, structural studies, and dynamic studies, E. coli provides a cost-effective system for producing isotope-enriched RCL. It should also be potential for producing other (2)H, (13)C, (15)N-enriched, Ile (δ1), Leu, Val methyl-protonated eukaryotic proteins with native disulfide bonds.
Asunto(s)
Lipasa/química , Lipasa/metabolismo , Rhizopus/enzimología , Isótopos de Carbono/metabolismo , Deuterio/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosilación , Marcaje Isotópico , Lipasa/genética , Isótopos de Nitrógeno/metabolismo , Pichia/genética , Pichia/metabolismo , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhizopus/químicaRESUMEN
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.
Asunto(s)
Integrasas/química , Virus de la Leucemia Murina/genética , Proteínas de Unión al ARN/química , Proteínas Virales/química , Integración Viral , Secuencia de Aminoácidos , Sitios de Unión , Islas de CpG , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Análisis de Secuencia de ADN , Eliminación de Secuencia , Factores de Transcripción , Sitio de Iniciación de la TranscripciónRESUMEN
Salicylic acid (SA) and its derivatives have been used for millennia to reduce pain, fever and inflammation. In addition, prophylactic use of acetylsalicylic acid, commonly known as aspirin, reduces the risk of heart attack, stroke and certain cancers. Because aspirin is rapidly de-acetylated by esterases in human plasma, much of aspirin's bioactivity can be attributed to its primary metabolite, SA. Here we demonstrate that human high mobility group box 1 (HMGB1) is a novel SA-binding protein. SA-binding sites on HMGB1 were identified in the HMG-box domains by nuclear magnetic resonance (NMR) spectroscopic studies and confirmed by mutational analysis. Extracellular HMGB1 is a damage-associated molecular pattern molecule (DAMP), with multiple redox states. SA suppresses both the chemoattractant activity of fully reduced HMGB1 and the increased expression of proinflammatory cytokine genes and cyclooxygenase 2 (COX-2) induced by disulfide HMGB1. Natural and synthetic SA derivatives with greater potency for inhibition of HMGB1 were identified, providing proof-of-concept that new molecules with high efficacy against sterile inflammation are attainable. An HMGB1 protein mutated in one of the SA-binding sites identified by NMR chemical shift perturbation studies retained chemoattractant activity, but lost binding of and inhibition by SA and its derivatives, thereby firmly establishing that SA binding to HMGB1 directly suppresses its proinflammatory activities. Identification of HMGB1 as a pharmacological target of SA/aspirin provides new insights into the mechanisms of action of one of the world's longest and most used natural and synthetic drugs. It may also provide an explanation for the protective effects of low-dose aspirin usage.
Asunto(s)
Aspirina/farmacología , Proteína HMGB1/genética , Inflamación/genética , Ácido Salicílico/farmacología , Aspirina/química , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Proteína HMGB1/biosíntesis , Proteína HMGB1/química , Humanos , Inflamación/tratamiento farmacológico , Inflamación/patología , Mutación , Resonancia Magnética Nuclear Biomolecular , Ácido Salicílico/químicaRESUMEN
The Solute Carrier (SLC) superfamily of integral membrane proteins function to transport a wide array of solutes across the plasma and organelle membranes. SLC proteins also function as important drug transporters and as viral receptors. Despite being classified as a single superfamily, SLC proteins do not share a single common fold classification; however, most belong to multi-pass transmembrane helical protein fold families. SLC proteins populate different conformational states during the solute transport process, including outward open, intermediate (occluded), and inward open conformational states. For some SLC fold families this structural "flipping" corresponds to swapping between conformations of their N-terminal and C-terminal symmetry-related sub-structures. Conventional AlphaFold2 or Evolutionary Scale Modeling methods typically generate models for only one of these multiple conformational states of SLC proteins. Here we describe a fast and simple approach for modeling multiple conformational states of SLC proteins using a combined ESM - AF2 process. The resulting multi-state models are validated by comparison with sequence-based evolutionary co-variance data (ECs) that encode information about contacts present in the various conformational states adopted by the protein. We also explored the impact of mutations on conformational distributions of SLC proteins modeled by AlphaFold2 using both conventional and enhanced sampling methods. This approach for modeling conformational landscapes of pseudo-symmetric SLC proteins is demonstrated for several integral membrane protein transporters, including SLC35F2 the receptor of a feline leukemia virus envelope protein required for viral entry into eukaryotic cells.
RESUMEN
Protein domain family PF06855 (DUF1250) is a family of small domains of unknown function found only in bacteria, and mostly in the order Bacillales and Lactobacillales. Here we describe the solution NMR or X-ray crystal structures of three representatives of this domain family, MW0776 and MW1311 from Staphyloccocus aureus and yozE from Bacillus subtilis. All three proteins adopt a four-helix motif similar to sterile alpha motif (SAM) domains. Phylogenetic analysis classifies MW1311 and yozE as functionally equivalent proteins of the UPF0346 family of unknown function, but excludes MW0776, which likely has a different biological function. Our structural characterization of the three domains supports this separation of function. The structures of MW0776, MW1311, and yozE constitute the first structural representatives from this protein domain family.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , Pliegue de Proteína , Staphylococcus aureus/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Bacillus subtilis/clasificación , Bacillus subtilis/genética , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Clonación Molecular , Cristalografía por Rayos X , Genes Bacterianos , Espectroscopía de Resonancia Magnética/métodos , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Especificidad de la Especie , Staphylococcus aureus/clasificación , Staphylococcus aureus/genética , Relación Estructura-ActividadRESUMEN
A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS(2)) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS(2)-tag is replaced with non-isotope labeled PrS(2)-tag, silencing the NMR signals from PrS(2)-tag in isotope-filtered (1)H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS(2)-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS(2) (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS(2)-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS(2)-tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone (1)H, (15)N and (13)C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear (1)H-(15)N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.
Asunto(s)
Marcaje Isotópico/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteína S/metabolismo , Proteínas/química , Proteínas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformación Proteica , Proteína S/genética , Proteínas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , SolubilidadRESUMEN
NMR is a valuable experimental tool in the structural biologist's toolkit to elucidate the structures, functions, and motions of biomolecules. The progress of machine learning, particularly in structural biology, reveals the critical importance of large, diverse, and reliable datasets in developing new methods and understanding in structural biology and science more broadly. Biomolecular NMR research groups produce large amounts of data, and there is renewed interest in organizing these data to train new, sophisticated machine learning architectures and to improve biomolecular NMR analysis pipelines. The foundational data type in NMR is the free-induction decay (FID). There are opportunities to build sophisticated machine learning methods to tackle long-standing problems in NMR data processing, resonance assignment, dynamics analysis, and structure determination using NMR FIDs. Our goal in this study is to provide a lightweight, broadly available tool for archiving FID data as it is generated at the spectrometer, and grow a new resource of FID data and associated metadata. This study presents a relational schema for storing and organizing the metadata items that describe an NMR sample and FID data, which we call Spectral Database (SpecDB). SpecDB is implemented in SQLite and includes a Python software library providing a command-line application to create, organize, query, backup, share, and maintain the database. This set of software tools and database schema allow users to store, organize, share, and learn from NMR time domain data. SpecDB is freely available under an open source license at https://github.rpi.edu/RPIBioinformatics/SpecDB.
Asunto(s)
Programas Informáticos , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
The conserved Lipoprotein-17 domain of membrane-associated protein Q9PRA0_UREPA from Ureaplasma parvum was selected for structure determination by the Northeast Structural Genomics Consortium, as part of the Protein Structure Initiative's program on structure-function analysis of protein domains from large domain sequence families lacking structural representatives. The 100-residue Lipoprotein-17 domain is a "domain of unknown function" (DUF) that is a member of Pfam protein family PF04200, a large domain family for which no members have characterized biochemical functions. The three-dimensional structure of the Lipoprotein-17 domain of protein Q9PRA0_UREPA was determined by both solution NMR and by X-ray crystallography at 2.5 Å. The two structures are in good agreement with each other. The domain structure features three α-helices, α1 through α3, and five ß-strands. Strands ß1/ß2, ß3/ß4, ß4/ß5 are anti-parallel to each other. Strands ß1and ß2 are orthogonal to strands ß3, ß4, ß5, while helix α3 is formed between the strands ß3 and ß4. One-turn helix α2 is formed between the strands ß1 and ß2, while helix α1 occurs in the N-terminal polypeptide segment. Searches of the Protein Data Bank do not identify any other protein with significant structural similarity to Lipoprotein-17 domain of Q9PRA0_UREPA, indicating that it is a novel protein fold.
Asunto(s)
Lipoproteínas/química , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular , Ureaplasma/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Mycoplasma/metabolismo , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , SolucionesRESUMEN
The extraterminal (ET) domain of BRD3 is conserved among BET proteins (BRD2, BRD3, BRD4), interacting with multiple host and viral protein-protein networks. Solution NMR structures of complexes formed between the BRD3 ET domain and either the 79-residue murine leukemia virus integrase (IN) C-terminal domain (IN329-408) or its 22-residue IN tail peptide (IN386-407) alone reveal similar intermolecular three-stranded ß-sheet formations. 15N relaxation studies reveal a 10-residue linker region (IN379-388) tethering the SH3 domain (IN329-378) to the ET-binding motif (IN389-405):ET complex. This linker has restricted flexibility, affecting its potential range of orientations in the IN:nucleosome complex. The complex of the ET-binding peptide of the host NSD3 protein (NSD3148-184) and the BRD3 ET domain includes a similar three-stranded ß-sheet interaction, but the orientation of the ß hairpin is flipped compared with the two IN:ET complexes. These studies expand our understanding of molecular recognition polymorphism in complexes of ET-binding motifs with viral and host proteins.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/química , Integrasas/química , Virus de la Leucemia Murina/enzimología , Proteínas Nucleares/química , Factores de Transcripción/química , Sitios de Unión , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Integrasas/metabolismo , Modelos Moleculares , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT((R)) SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354-359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two (15)N-, (13)C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline.
Asunto(s)
Clonación Molecular/métodos , Vectores Genéticos , Proteínas Recombinantes/biosíntesis , Sistema Libre de Células , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Ingeniería de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteómica/métodosRESUMEN
We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (>97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as>26,000 constructs. Over the past 10 years, more than 16,000 of these expressed protein, and more than 4400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last 5 years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.
Asunto(s)
Genómica/métodos , Proteínas/metabolismo , Proteómica/métodos , Clonación Molecular , Bases de Datos de Proteínas , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Espectroscopía de Resonancia Magnética , Proteínas/química , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30-200 microg in 8-35 microl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure.