RESUMO
Most G-protein-coupled receptors (GPCRs) are stabilized in common in the inactive state by the formation of the sodium ion-centered water cluster with the conserved Asp2.50 inside the seven-transmembrane domain. We determined the crystal structure of the leukotriene B4 (LTB4) receptor BLT1 bound with BIIL260, a chemical bearing a benzamidine moiety. Surprisingly, the amidine group occupies the sodium ion and water locations, interacts with D662.50, and mimics the entire sodium ion-centered water cluster. Thus, BLT1 is fixed in the inactive state, and the transmembrane helices cannot change their conformations to form the active state. Moreover, the benzamidine molecule alone serves as a negative allosteric modulator for BLT1. As the residues involved in the benzamidine binding are widely conserved among GPCRs, the unprecedented inverse-agonist mechanism by the benzamidine moiety could be adapted to other GPCRs. Consequently, the present structure will enable the rational development of inverse agonists specific for each GPCR.
Assuntos
Receptores do Leucotrieno B4/química , Sítio Alostérico , Amidinas/química , Animais , Ácido Aspártico/química , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Cobaias , Células HEK293 , Humanos , Fosfatos de Inositol/química , Leucotrieno B4/química , Ligantes , Ligação Proteica , Domínios Proteicos , Fator de Crescimento Transformador alfa/metabolismoRESUMO
The X-ray crystal structure of an anti-leukotriene (LT) C4 monoclonal antibody (mAbLTC) in complex with LTC4 was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4 single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4 which was comparable to that to LTD4 Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4 The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4 was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.
Assuntos
Afinidade de Anticorpos/genética , Leucotrieno E4/química , Mutação de Sentido Incorreto , Anticorpos de Cadeia Única/química , Substituição de Aminoácidos , Animais , Camundongos , Anticorpos de Cadeia Única/genéticaRESUMO
Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ', a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ' subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ' were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.
Assuntos
Complexo de Proteína do Fotossistema II/química , Proteínas de Plantas/química , Rodófitas/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Complexo de Proteína do Fotossistema II/ultraestrutura , Proteínas de Plantas/ultraestrutura , Conformação Proteica , Multimerização Proteica , Alinhamento de SequênciaRESUMO
OXA-58 is a class D ß-lactamase from the multi-drug resistant Acinetobacter baumannii. We determined the crystal structure of OXA-58 in a novel crystal, and revealed the structure of the substrate-binding cleft in a closed state, distinct from a previously reported OXA-58 crystal structure with the binding cleft in an open state. In the closed state, the movement of three loops (α3-α4, ß6-ß7, and ß8-α10) forms an arch-like architecture over the binding cleft through interaction between the Phe113 residues of α3-α4 and Met225 of ß6-ß7. This structure suggests the involvement of these flexible loops in OXA-58 substrate binding. In contrast to the mobile loops, the Ω-loop appeared static, including the conserved loop residues and their hydrogen bonds; the pivotal residue Trp169 within the Ω-loop, ζ-carbamic acid of the modified base catalyst residue Lys86, and nucleophilic residue Ser83. The stability of OXA-58 was enhanced concomitant with an increase in the hydrolytic activity catalyzed by NaHCO3-dependent ζ-carbamic acid formation, with an EC50 of 0.34 mM. The W169A mutant enzyme was significantly thermally unstable even in the presence of 100 mM NaHCO3, whereas the S83A mutant was stabilized with NaHCO3-dependent activation. The ζ-carbamic acid was shown to increase not only OXA-58 hydrolytic activity but also OXA-58 stability through the formation of a hydrogen bond network connected to the Ω-loop with Ser83 and Trp169. Thus, the static Ω-loop is important for OXA-58 stability, whereas the mobile loops of the substrate-binding cleft form the basis for accommodation of the various substituents of ß-lactam backbone.
Assuntos
beta-Lactamases/química , beta-Lactamases/metabolismo , beta-Lactamas/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Ligação de Hidrogênio , Hidrólise , Modelos Moleculares , Conformação ProteicaRESUMO
In this study, we introduced structure-based rational mutations in the guinea pig leukotriene B4 receptor (gpBLT1) in order to enhance the stabilization of the protein. Elements thought to be unfavorable for the stability of gpBLT1 were extracted based on the stabilization elements established in soluble proteins, determined crystal structures of G-protein-coupled receptors (GPCRs), and multiple sequence alignment. The two unfavorable residues His832.67 and Lys883.21, located at helix capping sites, were replaced with Gly (His83Gly2.67 and Lys88Gly3.21). The modified protein containing His83Gly2.67/Lys88Gly3.21 was highly expressed, solubilized, and purified and exhibited improved thermal stability by 4 °C in comparison with that of the original gpBLT1 construct. Owing to the double mutation, the expression level increased by 6-fold (Bmax=311 pmol/mg) in the membrane fraction of Pichia pastoris. The ligand binding affinity was similar to that of the original gpBLT1 without the mutations. Similar unfavorable residues have been observed at helix capping sites in many other GPCRs; therefore, the replacement of such residues with more favorable residues will improve stabilization of the GPCR structure for the crystallization.
RESUMO
BACKGROUND: Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC4 (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC). METHODS: We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC4 and LTD4 by competing their binding to their receptors. RESULTS: mAbLTC and scFvLTC inhibited their binding of LTC4 or LTD4 to CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R) overexpressed in Chinese hamster ovary cells. The induction by LTD4 of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT1R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC4- and LTD4-induced aggregation of mouse platelets expressing CysLT2R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT2R antagonists but not to CysLT1R antagonists. CONCLUSIONS: These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT1R and CysLT2R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors. GENERAL SIGNIFICANCE: mAbLTC can be used in the treatment of inflammatory diseases such as asthma.
Assuntos
Anticorpos Monoclonais/farmacologia , Leucotrieno C4/imunologia , Leucotrieno D4/imunologia , Anticorpos de Cadeia Única/farmacologia , Animais , Anticorpos Monoclonais/uso terapêutico , Asma/tratamento farmacológico , Células CHO , Cricetinae , Cricetulus , Citocinas/biossíntese , Humanos , Antagonistas de Leucotrienos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Agregação Plaquetária/efeitos dos fármacos , Receptores de Leucotrienos/efeitos dos fármacos , Receptores de Leucotrienos/fisiologiaRESUMO
The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4) and its metabolites, LTD4 and LTE4, are proinflammatory lipid mediators in asthma and other inflammatory diseases. They are generated through the 5-lipoxygenase/LTC4 synthase (LTC4S) pathway and act via at least two distinct G protein-coupled receptors. The inhibition of human LTC4S will make a simple way to treat the cys-LT relevant inflammatory diseases. Here, we show that compounds having 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid moiety suppress LTC4 synthesis, glutathione conjugation to the precursor LTA4, in both an enzyme assay and a whole-cell assay. Hierarchical in silico screenings of 6 million compounds provided 300,000 dataset for docking, and after energy minimization based on the crystal structure of LTC4S, 111 compounds were selected as candidates for a competitive inhibitor to glutathione. One of those compounds showed significant inhibitory activity, and subsequently, its derivative 5-((Z)-5-((E)-2-methyl-3-phenylallylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid (compound 1) was found to be the most potent inhibitor. The enzyme assay showed the IC50 was 1.9 µM and the corresponding 95% confidence interval was from 1.7 to 2.2 µM. The whole-cell assay showed that compound 1 was cell permeable and inhibited LTC4 synthesis in a concentration dependent manner.
Assuntos
Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Ácidos Ftálicos/farmacologia , Inibidores Enzimáticos/química , Estrutura Molecular , Ácidos Ftálicos/químicaRESUMO
Dodecyl-ß-D-selenomaltoside (SeDDM) is a seleno-detergent with a ß-glycosidic seleno-ether in place of the ether moiety in dodecyl-ß-D-maltoside. Seleno-detergents are candidates for heavy-atom agents in experimental phasing of membrane proteins in protein crystallography. Crystals of a nuclear membrane-embedded enzyme, leukotriene C(4) synthase (LTC(4)S), in complex with SeDDM were prepared and a multiwavelength anomalous diffraction (MAD) experiment was performed. The SeDDM in the LTC(4)S crystal exhibited sufficient anomalous diffraction for determination of the structure using MAD phasing.
Assuntos
Cristalografia por Raios X/métodos , Dissacarídeos/química , Glutationa Transferase/análise , Compostos de Organossilício/química , Animais , Glutationa Transferase/química , Humanos , Modelos Moleculares , Ligação ProteicaRESUMO
Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).
Assuntos
Arginina/química , Glutationa Transferase/química , Glutationa/química , Leucotrieno C4/química , Arginina/genética , Arginina/metabolismo , Asma/enzimologia , Asma/genética , Sítios de Ligação , Cristalografia por Raios X , Glutationa/genética , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Leucotrieno C4/biossíntese , Leucotrieno C4/genética , Mutação , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
There are hundreds of membrane protein atomic coordinates in the Protein Data Bank (PDB), and high-resolution structures of better than 2.5 A enable the visualization of a sizable number of amphiphiles (lipid and/or detergent) and bound water molecules as essential parts of the structure. Upon scrutinizing these high-resolution structures, water molecules were found to 'wedge' and stabilize large kink angle (30-40 degrees) in a simple cylindrical model at the transmembrane helical kinks so as to form an inter-helical cavity to accommodate a ligand binding or active site as a crucial structural feature in alpha-helical integral membrane proteins. Furthermore, some of these water molecules are proposed to play a pivotal role of their conformational change to exert their functional regulation.
Assuntos
Membrana Celular , Proteínas de Membrana/química , Água/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
The high yield expression of BLT1, a G-protein coupled receptor for leukotriene B(4), was established in Pichia pastoris for structural studies. Guinea pig BLT1 was expressed in a functional form without post-translational modifications for the rapid purification and the crystallization. Among the BLT1s from four species, only guinea pig BLT1 was successfully expressed with the comparable binding affinity to BLT1 of native guinea pig tissues for several ligands. Only Asn4 of the two putative N-glycosylation sites was glycosylated, and the mutation to Ala to avoid glycosylation did not affect the ligand binding affinity. However, the N-terminal region of the mutant was digested at the carboxyl ends of Arg3 and Arg8, as detected by N-terminal amino acid sequencing, and Ser309 in the C-terminal region was partially phosphorylated, as identified in the micro-sequencing by Q-TOF-MS/MS. To avoid chemical heterogeneity, the N-terminal peptide (1-14) truncated and the C-terminal phosphorylation-site eliminated mutant was generated. The binding affinity of the mutant's membrane fraction for LTB(4) was K(d)=6.6 nM and B(max)=50.0 pmol/mg membrane protein. The yield of purified mutant was approximately 0.3-0.4 mg from 1L culture, and the protein showed a single peak at molecular weight of 100 kDa in gel-filtration and no glycosylation or phosphorylation in MALDI-TOF MS.
Assuntos
Expressão Gênica , Pichia/genética , Receptores do Leucotrieno B4/genética , Receptores do Leucotrieno B4/isolamento & purificação , Sequência de Aminoácidos , Animais , Glicosilação , Cobaias , Dados de Sequência Molecular , Mutação , Fosforilação , Ligação Proteica , Receptores do Leucotrieno B4/química , Receptores do Leucotrieno B4/metabolismo , Regulação para CimaRESUMO
Leukotriene C(4) (LTC(4)) is synthesized by binding of glutathione to LTA(4), an epoxide derived from arachidonic acid, and further metabolized to LTD(4) and LTE(4). We previously prepared a monoclonal antibody with a high affinity and specificity to LTC(4). To explore the structure of the antigen-binding site of a monoclonal antibody against LTC(4) (mAbLTC), we isolated full-length cDNAs for heavy and light chains of mAbLTC. The heavy and light chains consisted of 461 and 238 amino acids including a signal peptide with molecular weights of 51,089 and 26,340, respectively. An expression plasmid encoding a single-chain antibody comprising variable regions of mAbLTC heavy and light chains (scFvLTC) was constructed and expressed in COS-7 cells. The recombinant scFvLTC showed a high affinity with LTC(4) comparable to mAbLTC. The scFvLTC also bound to LTD(4) and LTE(4) with 48% and 17% reactivities, respectively, as compared with LTC(4) binding, whereas the antibody showed almost no affinity for LTB(4).
Assuntos
Anticorpos Monoclonais/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Leucotrieno C4/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Clonagem Molecular , DNA Complementar/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
The extended-spectrum beta-lactamases are associated with antibiotic resistance. Toho-1 R274N/R276N, a Class A beta-lactamase of CTX-M-type, efficiently hydrolyzes first generationcephalosporins (for example, cephalothin), in addition to cefotaxime, a third generation cephalosporin. However, this enzyme only marginally hydrolyzes the third generation cephalosporin ceftazidime, and the monobactam aztreonam. The deacylation defectiveness of the mutant Toho-1 E166A/R274N/R276N, which lacks the deacylation activity, results in the accumulation of the complex of an acylated-enzyme intermediate analog. For drug design, it would be useful if a quantitative prediction of a catalytic property were available without the need of enzymatic measurements. Therefore, we examined whether there is a correlation between the thermal stability of a catalytic intermediate (analog) and its kinetic parameters. First we measured the hydrolytic kinetics of the 14 species of beta-lactam antibiotics by Toho-1 R274N/R276N, and also measured the thermal stability of the accumulated acyl-intermediates of Toho-1 E166A/R274N/R276 by differential scanning calorimetry. Here we report the correlation of these parameters. The logarithm of the catalytic efficiency for Toho-1 R274N/R276N, log(k(cat)/K(m)) exhibited the best linear correlation with T(m,) which is the heat-denaturation temperature midpoint of the corresponding acylated complex of Toho-1 E166A/R274N/R276N. The correlation coefficient was 0.947, indicating that a relationship exists between the kinetic parameters and the stability of the intermediates. The results demonstrate a new method for investigating the catalytic properties of enzymes against any substrates, and a new approach to designing enzymes.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Acilação , Substituição de Aminoácidos , Aztreonam/metabolismo , Varredura Diferencial de Calorimetria , Catálise , Ceftazidima/metabolismo , Desenho de Fármacos , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , beta-Lactamases/genéticaRESUMO
There has been accumulating evidence for the involvement of retroviral integrase (IN) in the reverse transcription of viral RNA. We previously identified a host factor, survival motor neuron-interacting protein 1 (SIP1/Gemin2) that binds to human immunodeficiency virus type 1 (HIV-1) IN and supports HIV-1 infection apparently at reverse transcription step. Here, we demonstrated that HIV-1 IN together with SIP1 augments reverse transcriptase (RT) activity by enhancing the assembly of RT on viral RNA in vitro. Synthetic peptides corresponding to the binding motifs within IN that inhibited the IN-SIP1 interaction abrogated reverse transcription in vitro and in vivo. Furthermore, knockdown of SIP1 reduced intracellular stability and multimer formation of IN through proteasome-mediated degradation machinery. Taken together, SIP1 appears to stabilize functional multimer forms of IN, thereby promoting the assembly of IN and RT on viral RNA to allow efficient reverse transcription, which is a prerequisite for efficient HIV-1 infection.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , Integrase de HIV/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Transcrição Reversa , Motivos de Aminoácidos , Linhagem Celular , Humanos , Imunoprecipitação , Peptídeos/química , Plasmídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , Proteínas Recombinantes/químicaRESUMO
Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD2)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-beta chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the beta-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys65 residue and its network of hydrogen bonds with the polar residues Ser45, Thr67, and Ser81, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys65 residue is activated by its interaction with Ser45 and Thr67 and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.
Assuntos
Oxirredutases Intramoleculares/fisiologia , Lipocalinas/química , Peptídeos beta-Amiloides/metabolismo , Animais , Catálise , Cristalografia por Raios X/métodos , Cisteína/química , Escherichia coli/metabolismo , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/metabolismo , Lipocalinas/fisiologia , Camundongos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
Many G protein-coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8 (H8), at the proximal region of the C-terminal tail. However, the significance of this domain is not fully understood. Here, we demonstrate the requirement of H8 for the proper folding of GPCRs for passage through the quality control in the endoplasmic reticulum (ER). In the human leukotriene B(4) type-2 receptor (hBLT2), lack of H8 led to an accumulation of the receptor (hBLT2/DeltaH8) in the ER. Similar results were obtained in two representative human GPCRs, dopamine type-1 and lysophosphatidic acid type-2 receptors, which were engineered to lack H8. Treatment with the several ligands, which act as pharmacological chaperones, facilitated the surface expression of hBLT2/DeltaH8. The surface-trafficked hBLT2/DeltaH8 exhibited an agonist-evoked increase in Ca(2+), demonstrating that H8 is not critical for ligand binding and activation of coupled G proteins. Thus, these results suggest that the H8 region of hBLT2 plays an important role in transport-competent receptor folding.
Assuntos
Retículo Endoplasmático/metabolismo , Receptores do Leucotrieno B4/química , Sequência de Aminoácidos , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Células HeLa , Humanos , Dobramento de Proteína , Transporte Proteico , Receptores de Dopamina D1/metabolismo , Receptores do Leucotrieno B4/agonistas , Receptores do Leucotrieno B4/antagonistas & inibidores , Receptores do Leucotrieno B4/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismoRESUMO
Prostaglandin E(2) is a major lipid mediator that regulates diverse biological processes. To elucidate how prostaglandin E(2) is recognized specifically by its antibody, the Fab fragment of a monoclonal anti-prostaglandin E(2) antibody was prepared and its complex with prostaglandin E(2) was crystallized. The stable Fab-prostaglandin E(2) complex was prepared by gel-filtration chromatography. Crystals were obtained by the microbatch method at 277 K using polyethylene glycol 4000 as a precipitant. A diffraction data set was collected to 2.2 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.3, b = 81.8, c = 82.2 A. The asymmetric unit was suggested to contain one molecule of the Fab-prostaglandin E(2) complex, with a corresponding crystal volume per protein weight of 2.75 A(3) Da(-1).
Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/química , Dinoprostona , Fragmentos Fab das Imunoglobulinas/química , Animais , Anticorpos Monoclonais/imunologia , Cristalização , Cristalografia por Raios X , Dinoprostona/química , Dinoprostona/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Ligantes , Camundongos , Estrutura Molecular , Difração de Raios XRESUMO
PCaP1, a hydrophilic cation-binding protein, is bound to the plasma membrane in Arabidopsis thaliana. We focused on the physicochemical properties of PCaP1 to understand its uniqueness in terms of structure and binding of metal ions. On fluorescence analysis, PCaP1 showed a signal of structural change in the presence of Cu(2+). The near-UV CD spectra showed a marked change of PCaP1 in CuCl(2) solution. The far-UV CD spectra showed the presence of alpha-helices and the intrinsically unstructured region. However, addition of Cu(2+) gave no change in the far-UV CD spectra. These results indicate that Cu(2+) induced a change in the tertiary structure without changing the secondary structure. The protein was sensitive to proteinase in the presence of Cu(2+), supporting that Cu(2+) is involved in the structural change. The PCaP1 solution was titrated with CuCl(2) and the change in the fluorescence spectrum was monitored to characterize Cu(2+)-binding properties. The obtained values of K(d) for Cu(2+) and the ligand-binding number were 10 microM and six ions per molecule, respectively. These findings indicate that PCaP1 has a high Cu(2+)-binding capacity with a relatively high affinity. PCaP1 lacks cysteine and histidine residues. A large number of glutamate residues may be involved in the Cu(2+) binding.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cobre/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Dicroísmo Circular , Cinética , Proteínas de Membrana/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , TermodinâmicaRESUMO
It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.
Assuntos
Proteínas de Bactérias/química , Thermus thermophilus/química , Proteínas de Bactérias/isolamento & purificação , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Microfluídica , Conformação Proteica , Fatores de Tempo , Difração de Raios XRESUMO
The beta-ketoacyl-(acyl carrier protein) synthases (beta-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 A resolution. The crystal is orthorhombic, space group P2(1)2(1)2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 A, and contains one homodimer in the asymmetric unit. The subunits adopt the well known alpha-beta-alpha-beta-alpha thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ;open' conformation of the Phe396 side chain.