RESUMO
Streptococcus pneumoniae is a major infectious agent responsible for pneumonia, otitis media, sepsis and meningitis. Pneumococcal surface protein A (PspA) is a well-characterized virulence factor localized on the surface and a target for vaccine development. In this study, we screened a single-chain antibody variable fragment (scFv) using phage display from a human synthetic library to select a clone 2B11. Affinity (Kd) of 2B11 was measured to be 5 nM using biolayer interferometry. 2B11 exhibited a dose-dependent recognition of recombinant PspA with no cross-reactivity towards pneumococcal antigens. The epitope on PspA was defined to residues 231-242 by mutational analysis. Molecular docking analysis supported the experimentally determined epitope, suggesting that the helix spanning residues 231-242 can bind to 2B11 with residues in the CDR-H3 (complementarity determining region 3 in the heavy chain) actively participating in the molecular contacts. Comparison of 2B11 with a commercial PspA antibody revealed that 2B11 exhibited a better specificity towards recombinant PspA antigen. 2B11 was capable of detecting endogenous PspA from pneumococcal lysates with affinity similar to that of the commercial antibody. Our study provides a molecular tool for biosensors detecting pneumococcal diseases.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Streptococcus pneumoniae/imunologia , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Técnicas Biossensoriais , Imunoensaio , Simulação de Acoplamento Molecular , Ligação Proteica , Anticorpos de Cadeia Única/isolamento & purificação , Streptococcus pneumoniae/químicaRESUMO
Early diagnosis of infectious diseases is important for treatment; therefore, selective and rapid detection of pathogenic bacteria is essential for human health. We report a strategy for highly selective detection and rapid separation of pathogenic microorganisms using magnetic nanoparticle clusters. Our approach to develop probes for pathogenic bacteria, including Salmonella, is based on a theoretically optimized model for the size of clustered magnetic nanoparticles. The clusters were modified to provide enhanced aqueous solubility and versatile conjugation sites for antibody immobilization. The clusters with the desired magnetic property were then prepared at critical micelle concentration (CMC) by evaporation-induced self-assembly (EISA). Two different types of target-specific antibodies for H- and O-antigens were incorporated on the cluster surface for selective binding to biological compartments of the flagella and cell body, respectively. For the two different specific binding properties, Salmonella were effectively captured with the O-antibody-coated polysorbate 80-coated magnetic nanoclusters (PCMNCs). The synergistic effect of combining selective targeting and the clustered magnetic probe leads to both selective and rapid detection of infectious pathogens.
Assuntos
Técnicas Bacteriológicas/métodos , Nanopartículas/química , Salmonella/isolamento & purificação , Anticorpos Antibacterianos/química , Técnicas Bacteriológicas/instrumentação , Espectroscopia de Ressonância Magnética , Magnetismo/instrumentação , Magnetismo/métodos , Polissorbatos/química , Salmonella/imunologia , Sorogrupo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Protein kinase CK2 is a ubiquitous kinase that can phosphorylate hundreds of cellular proteins and plays important roles in cell growth and development. Deregulation of CK2 is related to a variety of human cancers, and CK2 is regarded as a suppressor of apoptosis; therefore, it is a target of anticancer therapy. Nucleolar phosphoprotein 140 (Nopp140), which is an intrinsically disordered protein, interacts with CK2 and inhibits the latter's catalytic activity in vitro. Interestingly, the catalytic activity of CK2 is recovered in the presence of d-myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP6). IP6 is widely distributed in animal cells, but the molecular mechanisms that govern its cellular functions in animal cells have not been completely elucidated. In this study, the crystal structure of CK2 in complex with IP6 showed that the lysine-rich cluster of CK2 plays an important role in binding to IP6. The biochemical experiments revealed that a Nopp140 fragment (residues 568-596) and IP6 competitively bind to the catalytic subunit of CK2 (CK2α), and phospho-Ser574 of Nopp140 significantly enhances its interaction with CK2α. Substitutions of K74E, K76E, and K77E in CK2α significantly reduced the interactions of CK2α with both IP6 and the Nopp140-derived peptide. Our study gives an insight into the regulation of CK2. In particular, our work suggests that CK2 activity is inhibited by Nopp140 and reactivated by IP6 by competitive binding at the substrate recognition site of CK2.
Assuntos
Caseína Quinase II/química , Caseína Quinase II/metabolismo , Regulação da Expressão Gênica/fisiologia , Substâncias Macromoleculares/química , Modelos Moleculares , Proteínas Nucleares/química , Fosfoproteínas/química , Ácido Fítico/química , Substituição de Aminoácidos , Cristalização , Humanos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ácido Fítico/metabolismo , Conformação Proteica , Difração de Raios XRESUMO
When the Z-type variant of human α(1)-antitrypsin was overexpressed in Saccharomyces cerevisiae, proteomics analysis identified YLR301w as one of the up-regulated proteins. YLR301w is a 27.5 kDa protein with no sequence homology to any known protein and has been reported to interact with Sec72 and Hrr25. The crystal structure of S. cerevisiae YLR301w has been determined at 2.3 Å resolution, revealing a novel ß-structure. It consists of an N-terminal ten-stranded ß-barrel with two short α-helices connected by a 23-residue linker to a seven-stranded half-barrel with two short helices at the C-terminus. The N-terminal barrel has a highly conserved hydrophobic channel that can bind hydrophobic molecules such as PEG. It forms a homodimer both in the crystal and in solution. YLR301w binds Sec72 with a K(d) of 6.2 µM, but the biological significance of this binding requires further investigation.
Assuntos
Proteínas de Transporte/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Polietilenoglicóis/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
We introduce a simple and easy way to functionalize the surface of various carbonaceous materials through the ultraviolet light/ozone (UV/O3) plasma where we utilize the zero-, one-, and two-dimensional carbon frameworks. In a general manner, the lamps of a UV/O3 generator create two different wavelengths (λ = 185 and 254 nm); the shorter wavelength (λ = 185 nm) dissociates the oxygen (O2) in air and the longer wavelength (λ = 254 nm) dissociates the O3 and creates the reactive and monoatomic oxygen radical, which tends to incorporate onto the defects of the carbons. By tailoring the association and dissociation of the oxygen with various forms, carbon black, carbon nanofibers, and graphite flakes, chosen as representative models for the zero-, one-, and two-dimensional carbon frameworks, their structure can be oxidized, respectively, which is known as photochemical oxidation. Various carbons have their own distinctive morphology and electron transport properties, which are applicable for the lithium-sulfur (Li-S) cell. We, here, report on the improvement of electrochemical performance of the lithium/sulfur cell through such an efficient functionalization approach.
RESUMO
Enoyl-[acyl carrier protein] reductase (ENR) is an essential enzyme in type II fatty-acid synthesis that catalyzes the last step in each elongation cycle. Thus far FabI, FabL and FabK have been reported to carry out the reaction, with FabI being the most characterized. Some bacteria have more than one ENR, and Bacillus cereus has two (FabI and FabL) reported. Here, we have determined the crystal structures of the later in the apo form and in the ternary complex with NADP(+) and an indole naphthyridinone inhibitor. The two structures are almost identical, except for the three stretches that are disordered in the apo form. The apo form exists as a homo-dimer in both crystal and solution, while the ternary complex forms a homo-tetramer. The three stretches disordered in the apo structure are important in the cofactor and the inhibitor binding as well as in tetramer formation.
Assuntos
Bacillus cereus/enzimologia , Enoil-(Proteína de Transporte de Acila) Redutase (NADPH, B-Específica)/química , Sequência de Aminoácidos , Apoenzimas/química , Cristalografia por Raios X , Enoil-(Proteína de Transporte de Acila) Redutase (NADPH, B-Específica)/antagonistas & inibidores , Inibidores Enzimáticos/química , Dados de Sequência Molecular , NADP/química , Multimerização Proteica , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Farnesoid X receptor (FXR) serves as a receptor for chenodeoxycholic acid (CDCA) and other bile acids, and it coordinates cholesterol and lipid metabolism. Because targeting the FXR-CDCA interaction might provide a way to regulate lipid homeostasis, we developed an FXR binding assay based on fluorescence polarization. Employing a fluorescently labeled CDCA (CDCA-F), we showed that CDCA-F selectively bound to the ligand binding domain of FXR (FXR-LBD) among nuclear receptors. The assay was then used for screening inhibitors against the FXR-CDCA interaction, thereby discovering four relatively potent inhibitors. The selected inhibitors were further studied for changes in intrinsic tryptophan fluorescence of FXR-LBD to gain structural insights into the interaction. Furthermore, transactivation effects of the inhibitors on the human bile salt excretory pump (BSEP) promoter were examined to reveal their cellular activities in the FXR-mediated pathway. Therefore, we demonstrated that the developed assay would offer an efficient primary screening tool for identifying FXR modulators.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Polarização de Fluorescência , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Ácido Quenodesoxicólico/metabolismo , Corantes Fluorescentes/metabolismo , Genes Reporter , Humanos , Ligantes , Camundongos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Espectrometria de Fluorescência , Especificidade por Substrato , Ativação TranscricionalRESUMO
By simple pyrolysis of a tin salen complex [Sn(salen)] and sulfur powder at 700 °C, SnS nanoparticles with â¼20 nm thickness homogeneously embedded in nitrogen-doped carbon are prepared. When applied as lithium-ion battery anodes, the SnS/N-C nanocomposites exhibited long cycling stability and excellent rate capability.
RESUMO
PDF (peptide deformylase) plays a critical role in the production of mature proteins by removing the N-formyl polypeptide of nascent proteins in the prokaryote cell system. This protein is essential for bacterial growth, making it an attractive target for the design of new antibiotics. Accordingly, PDF has been evaluated as a drug target; however, architectural mechanism studies of PDF have not yet fully elucidated its molecular function. We recently reported the crystal structure of PDF produced by Enterococcus faecium [K.H. Nam, J.I. Ham, A. Priyadarshi, E.E. Kim, N. Chung, K.Y. Hwang, "Insight into the antibacterial drug design and architectural mechanism of peptide recognition from the E. faecium peptide deformylase structure", Proteins 74 (2009) 261-265]. Here, we present the crystal structure of the EfPDF complex with MAS (Met-Ser-Ala), thereby not only delineating the architectural mechanism for the recognition of mimic-peptides by N-terminal cleaved expression peptide, but also suggesting possible targets for rational design of antibacterial drugs. In addition to their implications for drug design, these structural studies will facilitate elucidation of the architectural mechanism responsible for the peptide recognition of PDF.
Assuntos
Amidoidrolases/química , Enterococcus faecium/enzimologia , Oligopeptídeos/química , Amidoidrolases/genética , Cristalografia por Raios X , Desenho de Fármacos , Conformação ProteicaRESUMO
D-Glutamic acid is a required biosynthetic building block for peptidoglycan, and the enzyme glutamate racemase (GluR) catalyzes the inter-conversion of D and L-glutamate enantiomers. Therefore, GluR is considered as an attractive target for the design of new antibacterial drugs. Here, we report the crystal structures of GluR from Streptococcus pyogenes in both inhibitor-free and inhibitor-bound forms. The inhibitor free GluR crystallized in two different forms, which diffracted to 2.25 A and 2.5 A resolution, while the inhibitor-bound crystal diffracted to 2.5 A resolution. GluR is composed of two domains of alpha/beta protein that are related by pseudo-2-fold symmetry and the active site is located at the domain interface. The inhibitor, gamma-2-naphthylmethyl-D-glutamate, which was reported earlier as a novel potent competitive inhibitor, makes several hydrogen bonds with protein atoms, and the naphthyl moiety is located in the hydrophobic pocket. The inhibitor binding induces a disorder in one of the loops near the active site. In both crystal forms, GluR exists as a dimer and the interactions seen at the dimer interface are almost identical. This agrees well with the results from gel filtration and dynamic light-scattering studies.
Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Isomerases de Aminoácido/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Streptococcus pyogenes/enzimologia , Isomerases de Aminoácido/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Inibidores Enzimáticos/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The nickel and cobalt resistance of Cupriavidus metallidurans CH34 is mediated by the CnrCBA efflux pump encoded by the cnrYHXCBAT metal resistance determinant. The products of the three genes cnrYXH transcriptionally regulate expression of cnr. CnrY and CnrX are membranebound proteins, probably functioning as anti-sigma factors, whereas CnrH is a cnr-specific extracytoplasmic functions (ECF) sigma factor. The periplasmic domain of CnrX (residues 29- 148) was cloned as a N-terminal His-tagged protein, expressed in Escherichia coli, and purified using affinity chromatography and gel filtration. The molecular mass was estimated to be about 13.6 kDa by size exclusion chromatography, corresponding to a monomer. The tetragonal bipyramid crystals were obtained by mixing an equal volume of protein in 50 mM Tris-HCl, pH 7.5, 1% glycerol, 100 mM NaCl, 1 mM DTT, and the reservoir solution of 15% w/v PEG 2000, 100 mM lithium chloride at 277 K in 2-4 days using hanging drop vapor diffusion. The protein concentration was 24 mg/ml. The crystal that diffracted to 2.42 A resolution belongs to space group P41 or P4(3) with unit cell parameters of a=b=32.14 A, c=195.31 A, alpha=beta=gamma=90 degrees, with one molecule of CnrX in the asymmetric unit.
Assuntos
Proteínas de Bactérias , Cobalto/farmacologia , Cupriavidus/efeitos dos fármacos , Farmacorresistência Bacteriana , Níquel/farmacologia , Periplasma/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Cupriavidus/genética , Cupriavidus/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Peptide deformylase (PDF) is a metalloenzyme that removes the N-terminal formyl groups from newly synthesized proteins. It is essential for bacterial survival, and is therefore-considered as a potential target for antimicrobial chemotherapy. However, some bacteria including medically relevant pathogens possess two or more def-like genes. Here we have examined two PDFs from Bacillus cereus. The two share only 32% sequence identity and the crystal structures show overall similarity with PDF2 having a longer C-terminus. However, there are differences at the two active sites, and these differences appear to contribute to the activity difference seen between the two. BcPDF2 is found as a dimer in the crystal form with two additional actinonin bound at that interface.
Assuntos
Amidoidrolases/química , Bacillus cereus/enzimologia , Amidoidrolases/genética , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Bacillus cereus/genética , Sequência de Bases , Domínio Catalítico , Cristalografia por Raios X , Primers do DNA/genética , DNA Bacteriano/genética , Dimerização , Genes Bacterianos , Ácidos Hidroxâmicos/química , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
Enoyl-[acyl-carrier protein] reductase (enoyl-ACP reductase; ENR) is a key enzyme in type II fatty-acid synthase that catalyzes the last step in each elongation cycle. It has been considered as an antibiotic target since it is an essential enzyme in bacteria. However, recent studies indicate that some pathogens have more than one ENR. Bacillus subtilis is reported to have two ENRs, namely BsFabI and BsFabL. While BsFabI is similar to other FabIs, BsFabL shows very little sequence similarity and is NADPH-dependent instead of NADH-dependent as in the case of FabI. In order to understand these differences on a structural basis, BsFabL has been cloned, expressed and and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 A, alpha = beta = 90, gamma = 120 degrees and one molecule of FabL in the asymmetric unit. Data were collected using synchrotron radiation (beamline 4A at the Pohang Light Source, Korea). The crystal diffracted to 2.5 A resolution.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Cristalografia por Raios X/métodos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Proteínas de Bactérias/isolamento & purificação , Cristalização , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/isolamento & purificaçãoRESUMO
Peptide assemblies have received significant attention because of their important role in biology and applications in bionanotechnology. Despite recent efforts to elucidate the principles of peptide self-assembly for developing novel functional devices, peptide self-assembly on two-dimensional nanomaterials has remained challenging. Here, we report nature-inspired two-dimensional peptide self-assembly on pristine graphene via optimization of peptide-peptide and peptide-graphene interactions. Two-dimensional peptide self-assembly was designed based on statistical analyses of >104 protein structures existing in nature and atomistic simulation-based structure predictions. We characterized the structures and surface properties of the self-assembled peptide formed on pristine graphene. Our study provides insights into the formation of peptide assemblies coupled with two-dimensional nanomaterials for further development of nanobiocomposite devices.
RESUMO
RanBPM and RanBP10 are non-canonical members of the Ran binding protein family that lack the Ran binding domain and do not associate with Ran GTPase in vivo. Rather, they have been shown to be scaffolding proteins that are important for a variety of cellular processes, and both of these proteins contain a SPRY domain, which has been implicated in mediating protein-protein interactions with a variety of targets including the DEAD-box containing ATP-dependent RNA helicase (DDX-4). In this study, we have determined the crystal structures of the SPIa and the ryanodine receptor domain and of approximately 70 upstream residues (immediate upstream to SPRY motif) of both RanBPM and RanBP10. They are almost identical, composed of a ß-sandwich fold with a set of two helices on each side located at the edge of the sheets. A unique shallow binding surface is formed by highly conserved loops on the surface of the ß-sheet with two aspartates on one end, a positive patch on the opposite end, and a tryptophan lining at the bottom of the surface. The 20-mer peptide (residues 228-247) of human DDX-4, an ATP-dependent RNA helicase known to regulate germ cell development, binds to this surface with a KD of ~13µM. The crystal structure of the peptide complex and the mutagenesis studies elucidate how RanBPM can recognize its interaction partners to function in gametogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Domínio B30.2-SPRY , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Cristalografia por Raios X , Proteínas do Citoesqueleto/genética , Células Germinativas/fisiologia , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Proteínas Nucleares/genética , Ligação Proteica , Conformação ProteicaRESUMO
Learning to engineer self-assembly would enable the precise organization of molecules by design to create matter with tailored properties. Here we demonstrate that proteins can direct the self-assembly of buckminsterfullerene (C60) into ordered superstructures. A previously engineered tetrameric helical bundle binds C60 in solution, rendering it water soluble. Two tetramers associate with one C60, promoting further organization revealed in a 1.67-Å crystal structure. Fullerene groups occupy periodic lattice sites, sandwiched between two Tyr residues from adjacent tetramers. Strikingly, the assembly exhibits high charge conductance, whereas both the protein-alone crystal and amorphous C60 are electrically insulating. The affinity of C60 for its crystal-binding site is estimated to be in the nanomolar range, with lattices of known protein crystals geometrically compatible with incorporating the motif. Taken together, these findings suggest a new means of organizing fullerene molecules into a rich variety of lattices to generate new properties by design.
Assuntos
Fulerenos/química , Peptídeos/química , Multimerização Proteica , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dados de Sequência Molecular , Peptídeos/síntese química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Soluções , Eletricidade EstáticaRESUMO
Enoyl-[acyl carrier protein] (ACP) reductase (ENR) is a key enzyme in type II fatty acid synthesis that catalyzes the last step in each elongation cycle. Therefore, it has been considered as a target for antibiotics. However, recent studies indicate that some pathogens have more than one ENR; in particular, Bacillus subtilis has two ENRs, FabI and FabL. The crystal structures of the ternary complexes of BsFaBI and BsFabL are found as a homotetramer showing the same overall structure despite a sequence identity of only 24%. The positions of the catalytic dyad of Tyr-(Xaa)(6)-Lys in FabL are almost identical to that of FabI, but a detailed structural analysis shows that FabL shares more structural similarities with FabG and other members of the SDR (short-chain alcohol dehydrogenase/reductase) family. The apo FabL structure shows significantly different conformations at the cofactor and the substrate-binding regions, and this resulted in a totally different tetrameric arrangement reflecting the flexibility of these regions in the absence of the cofactor and substrate/inhibitor.