The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2 Å resolution, the highest yet reported. Faster-growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only ∼1.6 Å resolution and are not analysed further here. The 1.2 Å resolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen-bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near-atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out.

• MeSH
• difrakce rentgenového záření MeSH
• flavinadenindinukleotid chemie   metabolismus   MeSH
• flavinmononukleotid chemie   metabolismus   MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• NAD(P)H dehydrogenasa (chinon) chemie   metabolismus   MeSH
• oxidace-redukce MeSH
• proteiny z Escherichia coli chemie   metabolismus   MeSH
• represorové proteiny chemie   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Crystallography provides unique information about the arrangement of water molecules near protein surfaces. Using a nonredundant set of 2818 protein crystal structures with a resolution of better than 1.8 Å, the extent and structure of the hydration shell of all 20 standard amino-acid residues were analyzed as function of the residue conformation, secondary structure and solvent accessibility. The results show how hydration depends on the amino-acid conformation and the environment in which it occurs. After conformational clustering of individual residues, the density distribution of water molecules was compiled and the preferred hydration sites were determined as maxima in the pseudo-electron-density representation of water distributions. Many hydration sites interact with both main-chain and side-chain amino-acid atoms, and several occurrences of hydration sites with less canonical contacts, such as carbon-donor hydrogen bonds, OH-π interactions and off-plane interactions with aromatic heteroatoms, are also reported. Information about the location and relative importance of the empirically determined preferred hydration sites in proteins has applications in improving the current methods of hydration-site prediction in molecular replacement, ab initio protein structure prediction and the set-up of molecular-dynamics simulations.

• MeSH
• aminokyseliny analýza   MeSH
• databáze proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• proteiny chemie   MeSH
• sekundární struktura proteinů MeSH
• voda analýza   MeSH
• vodíková vazba MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Deltahedral metallacarborane compounds have recently been discovered as potent, specific, stable, and nontoxic inhibitors of HIV-1 protease (PR), the major target for AIDS therapy. The 2.15 A-resolution X-ray structure has exhibited a nonsymmetrical binding of the parental compound [Co(3+)-(C2B9H11)2](-) (GB-18) into PR dimer and a symmetrical arrangement in the crystal of two PR dimer complexes into a tetramer. In order to explore structural and energetic details of the inhibitor binding, quantum mechanics coupled with molecular mechanics approach was utilized. Realizing the close positioning of anionic inhibitors in the active site cavity, the possibility of an exchange of structural water molecules Wat50 and Wat128 by Na+ counterions was studied. The energy profiles for the rotation of the GB-18 molecules along their longitudinal axes in complex with PR were calculated. The results show that two Na+ counterions are present in the active site cavity and provide energetically favorable and unfavorable positions for carbon atoms within the carborane cages. Eighty-one rotamer combinations of four molecules of GB-18 bound to PR out of 4 x 10(5) are predicted to be highly populated. These results lay ground for further calculations of interaction energies between GB-18 and amino acids of PR active site and will make it possible to interpret computationally the binding of similar metallacarborane molecules to PR as well as to resistant PR variants. Moreover, this computational tool will allow the design of new, more potent metallacarborane-based HIV-1 protease inhibitors.

• MeSH
• financování organizované MeSH
• HIV-proteasa chemie   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• kvantová teorie MeSH
• ligandy MeSH
• molekulární struktura MeSH
• organokovové sloučeniny chemie   MeSH
• termodynamika MeSH

UNLABELLED: The Pden_2689 gene encoding FerA, an NADH:flavin oxidoreductase required for growth of Paracoccus denitrificans under iron limitation, was cloned and overexpressed as a C-terminally His6-tagged derivative. The binding of substrates and products was detected and quantified by isothermal titration calorimetry and fluorometric titration. FerA binds FMN and FAD with comparable affinity in an enthalpically driven, entropically opposed process. The reduced flavin is bound more loosely than the oxidized one, which was confirmed by a negative shift in the redox potential of FMN after addition of FerA. Initial velocity and substrate analogs inhibition studies showed that FerA follows a random-ordered sequence of substrate (NADH and FMN) binding. The primary kinetic isotope effects from stereospecifically deuterated nicotinamide nucleotides demonstrated that hydride transfer occurs from the pro-S position and contributes to rate limitation for the overall reaction. The crystal structure of FerA revealed a twisted seven-stranded antiparallel β-barrel similar to that of other short chain flavin reductases. Only minor structural changes around Arg106 took place upon FMN binding. The solution structure FerA derived from small angle X-ray scattering (SAXS) matched the dimer assembly predicted from the crystal structure. Site-directed mutagenesis pinpointed a role of Arg106 and His146 in binding of flavin and NADH, respectively. Pull down experiments performed with cytoplasmic extracts resulted in a negative outcome indicating that FerA might physiologically act without association with other proteins. Rapid kinetics experiments provided evidence for a stabilizing effect of another P. denitrificans protein, the NAD(P)H: acceptor oxidoreducase FerB, against spontaneous oxidation of the FerA-produced dihydroflavin.

• MeSH
• chromatografie afinitní MeSH
• exprese genu MeSH
• flavinadenindinukleotid metabolismus   MeSH
• flavinmononukleotid metabolismus   MeSH
• FMN-reduktasa chemie   genetika   metabolismus   MeSH
• kinetika MeSH
• klonování DNA MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• maloúhlový rozptyl MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• NAD metabolismus   MeSH
• Paracoccus denitrificans enzymologie   genetika   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH

Guanine-rich DNA has the potential to fold into non-canonical G-quadruplex (G4) structures. Analysis of the genome of the social amoeba Dictyostelium discoideum indicates a low number of sequences with G4-forming potential (249-1055). Therefore, D. discoideum is a perfect model organism to investigate the relationship between the presence of G4s and their biological functions. As a first step in this investigation, we crystallized the dGGGGGAGGGGTACAGGGGTACAGGGG sequence from the putative promoter region of two divergent genes in D. discoideum. According to the crystal structure, this sequence folds into a four-quartet intramolecular antiparallel G4 with two lateral and one diagonal loops. The G-quadruplex core is further stabilized by a G-C Watson-Crick base pair and a A-T-A triad and displays high thermal stability (Tm > 90°C at 100 mM KCl). Biophysical characterization of the native sequence and loop mutants suggests that the DNA adopts the same structure in solution and in crystalline form, and that loop interactions are important for the G4 stability but not for its folding. Four-tetrad G4 structures are sparse. Thus, our work advances understanding of the structural diversity of G-quadruplexes and yields coordinates for in silico drug screening programs and G4 predictive tools.

• MeSH
• cirkulární dichroismus MeSH
• Dictyostelium genetika   MeSH
• G-kvadruplexy * MeSH
• genom MeSH
• konformace nukleové kyseliny * MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• mutace MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• promotorové oblasti (genetika) MeSH
• spektrofotometrie ultrafialová MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• MeSH
• molekulární biologie MeSH
• proteiny MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie
• molekulární biologie, molekulární medicína

Isoalkyl (isoalkyl = isopropyl-(L1), isobutyl-(L2) and isoamyl-(L3)) derivatives of thiosalicylic acid (TSA) were prepared by alkylation of TSA with corresponding isoalkyl-chlorides in the alkaline water-ethanol solution. The new free copper(II)-complexes with corresponding S-isoalkyl derivatives of TSA (C1-copper(II)-complex with S-isopropyl derivative of thiosalicylic acid, C2-copper(II)-complex with S-isobutyl derivative of thiosalicylic acid and C3-copper(II)-complex with S-isoamyl derivative of thiosalicylic acid) have been synthesized by direct reaction of copper(II)-nitrate with ligand precursor and then characterized by microanalysis, infrared spectra (IR) and EPR (electron paramagnetic resonance) spectra. The spectroscopically predicted structure of the obtained binuclear copper(II)-complex with S-isopropyl derivative of thiosalicylic acid was confirmed by X-ray analysis. Single crystals suitable for X-ray measurements were obtained by slow crystallization from a water solution. Newly synthesized precursors S-isoalkyl derivatives of thiosalicylic acid and corresponding copper(II)-complexes moderately reduced viability of human and murine lung cancer cells, they showed similar cytotoxic effect on human colorectal cancer cells as cisplatin and lower cytotoxic effect than cisplatin toward normal fibroblasts, evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric technique. All new complexes exhibited apoptotic effect toward lung cancer cells, stronger than cisplatin, whereas only C3 induced significant apoptosis of colorectal cancer cells. Complex C1 showed significant antiproliferative effect against murine lung cancer cells, LLC1, while C2 reduced expression of Ki67 in human colorectal cancer cells. All tested complexes induced cell cycle arrest of HCT116 cells in G2/M phase.

• MeSH
• buňky A549 MeSH
• cytotoxiny * chemická syntéza   chemie   farmakologie   MeSH
• komplexní sloučeniny * chemická syntéza   chemie   farmakologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• měď * chemie   farmakologie   MeSH
• molekulární struktura MeSH
• salicylany * chemická syntéza   chemie   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Non-isothermal differential scanning calorimetry was used to study the influences of particle size and mechanically induced defects on the recrystallization kinetics of amorphous Enzalutamide. Enzalutamide prepared by hot melt extrusion and spray-drying was used as a model material. The recrystallization rate was primarily accelerated by the presence of the processing-damaged surface of the powder particles. The actual surface/volume ratio associated with decreasing particle size fulfilled only a secondary role. Interestingly, higher quench rate during the extrusion led to a formation of thermally less stable material (with the worse stability being manifested via lower activation energy of crystal growth in the amorphous matrix). This can be the consequence of the formation of looser structure more prone to rearrangements. The recrystallization kinetics of the prepared Enzalutamide amorphous materials was described by the two-parameter autocatalytic kinetic model. The modified single-curve multivariate kinetic analysis (optimized for the data obtained at heating rate 0.5 °C•min-1) was used to calculate the extrapolated kinetic predictions of long-term isothermal crystal growth. The predictions were made for the temperatures from the range of drug shelf-life and processing for each particle size fraction. By the combination of the mass-weighted predictions for the individual powder fractions it was possible to obtain a very reasonable (temperature-extrapolated) prediction of the crystallization rate for the as-prepared unsieved powdered amorphous Enzalutamide.

• MeSH
• benzamidy MeSH
• diferenciální skenovací kalorimetrie MeSH
• fenylthiohydantoin MeSH
• kinetika MeSH
• krystalizace MeSH
• nitrily MeSH
• stabilita léku MeSH
• velikost částic MeSH
• vysoká teplota * MeSH
• Publikační typ
• časopisecké články MeSH

Lectins with a β-propeller fold bind glycans on the cell surface through multivalent binding sites and appropriate directionality. These proteins are formed by repeats of short domains, raising questions about evolutionary duplication. However, these repeats are difficult to detect in translated genomes and seldom correctly annotated in sequence databases. To address these issues, we defined the blade signature of the five types of β-propellers using 3D-structural data. With these templates, we predicted 3,887 β-propeller lectins in 1,889 species and organized this information in a searchable online database. The data reveal a widespread distribution of β-propeller lectins across species. Prediction also emphasizes multiple architectures and led to the discovery of a β-propeller assembly scenario. This was confirmed by producing and characterizing a predicted protein coded in the genome of Kordia zhangzhouensis. The crystal structure uncovers an intermediate in the evolution of β-propeller assembly and demonstrates the power of our tools.

• MeSH
• Archaea chemie   MeSH
• Bacteria chemie   MeSH
• databáze proteinů MeSH
• Eukaryota chemie   MeSH
• genom bakteriální MeSH
• lektiny chemie   MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• proteom MeSH
• sbalování proteinů MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

To find an effective drug for Zika virus, it is important to understand how numerous proteins which are critical for the virus' structure and function interact with their counterparts. One approach to inhibiting the flavivirus is to deter its ability to bind onto glycoproteins; however, the crystal structures of envelope proteins of the ever-evolving viral strains that decipher glycosidic or drug-molecular interactions are not always available. To fill this gap, we are reporting a holistic, simulation-based approach to predict compounds that will inhibit ligand binding onto a structurally unresolved protein, in this case the Zika virus envelope protein (ZVEP), by developing a three-dimensional general structure and analyzing sites at which ligands and small drug-like molecules interact. By examining how glycan molecules and small-molecule probes interact with a freshly resolved ZVEP homology model, we report the susceptibility of ZVEP to inhibition via two small molecules, ZINC33683341 and ZINC49605556-by preferentially binding onto the primary receptor responsible for the virus' virulence. Antiviral activity was confirmed when ZINC33683341 was tested in cell culture. We anticipate the results to be a starting point for drug discovery targeting Zika virus and other emerging pathogens.

• MeSH
• antivirové látky chemie   farmakologie   MeSH
• Cercopithecus aethiops MeSH
• knihovny malých molekul chemie   farmakologie   MeSH
• molekulární modely MeSH
• počítačová simulace MeSH
• polysacharidy metabolismus   MeSH
• proteiny virového obalu antagonisté a inhibitory   chemie   MeSH
• strukturní homologie proteinů MeSH
• vazebná místa MeSH
• Vero buňky MeSH
• virová nálož účinky léků   MeSH
• virus zika účinky léků   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH