Two types of entomocidal crystals of Bacillus thuringiensis ssp. finitimus have the same set of unique delta-endotoxins

The strain B-1166 differs from the other strains of Bacillus thuringiensis ssp. finitimus because it has two crystal types with different localization in the sporulating cell, i.e., inside and outside of exosporium membrane. Two dissociants of the strain were obtained containing only one of the crystal types. The initial strain produces at least three various delta-endotoxins (Fin2, Fin3, and Fin5) differing from all other known entomocidal proteins; Fin2 and Fin3 are similar to each other but differ from Fin5. Both crystal types contain the same endotoxins (Fin2, Fin3, and Fin5). In the B-1166 strain the site of crystal deposition is not determined by their protein composition.     

Functional characterization of 2',5'-linked oligoadenylate binding determinant of human RNase L

RNase L is activated by the binding of unusual 2',5'-linked oligoadenylates (2-5A) and acts as the effector enzyme of the 2-5A system, an interferon-induced anti-virus mechanism. Efforts have been made to understand the 2-5A binding mechanism, not only for scientific interests but also for the prospects that the understanding of such mechanisms lead to new remedies for viral diseases. We have recently elucidated the crystal structure of the 2-5A binding ankyrin repeat domain of human RNase L complexed with 2-5A. To determine the contributions of amino acid residues surrounding the 2-5A binding site, point mutants and a deletion mutant were designed based on the crystal structure. These mutant proteins were analyzed for their interaction with 2-5A using a steady-state fluorescence technique. In addition, full-length RNase L mutants were tested for their activation by 2-5A. The results reveal that pi-pi stacking interactions of Trp60 and Phe126, electrostatic interactions of Lys89 and Arg155, and hydrogen bonding by Glu131 make crucial contributions to 2-5A binding. It was also found that the crystal structure of the ankyrin repeat domain L.2-5A complex accurately portrays the 2-5A binding mode in full-length RNase L.     

Molecular conformation of ascidiacyclamide, a cytotoxic cyclic peptide from Ascidian: X-ray analyses of its free form and solvate crystals.

In order to investigate the conformational variation of ascidiacyclamide, a cytotoxic cyclic peptide from marine tunicate Ascidian, single crystals were prepared from ethanol and aqueous ethanol solutions as its free form (crystal I) and H2O/0.5 C2H5OH solvate (crystal II), respectively, and were determined by the x-ray diffraction method. Crystal I showed a pseudo C2-symmetric saddle-shaped rectangular conformation. Similar conformations were also observed in crystal II, where there were two crystallographically independent C2-symmetric molecules (named Mol-A and -B) per asymmetric unit. Mol-A and -B included H2O and H2O/C2H5OH solvents within their ring structures, respectively. These water and ethanol molecules were located on the crystallographic dyad axes, and were stabilized by the van der Waals contacts (including hydrogen bonds) with the polar-ring N atoms and nonpolar D-Val side-chain atoms. The conformational characteristics of ascidiacyclamide and its fluctuation/variation were discussed based on the present and previously reported x-ray results.     

Solution structure of C-terminal Escherichia coli translation initiation factor IF2 by small-angle X-ray scattering

Initiation of protein synthesis in bacteria involves the combined action of three translation initiation factors, including translation initiation factor IF2. Structural knowledge of this bacterial protein is scarce. A fragment consisting of the four C-terminal domains of IF2 from Escherichia coli was expressed, purified, and characterized by small-angle X-ray scattering (SAXS), and from the SAXS data, a radius of gyration of 43 +/- 1 A and a maximum dimension of approximately 145 A were obtained for the molecule. Furthermore, the SAXS data revealed that E. coli IF2 in solution adopts a structure that is significantly different from the crystal structure of orthologous aIF5B from Methanobacterium thermoautotrophicum. This crystal structure constitutes the only atomic resolution structural knowledge of the full-length factor. Computer programs were applied to the SAXS data to provide an initial structural model for IF2 in solution. The low-resolution nature of SAXS prevents the elucidation of a complete and detailed structure, but the resulting model for C-terminal E. coli IF2 indicates important structural differences between the aIF5B crystal structure and IF2 in solution. The chalice-like structure with a highly exposed alpha-helical stretch observed for the aIF5B crystal structure was not found in the structural model of IF2 in solution, in which domain VI-2 is moved closer to the rest of the protein.     

Chlorination of 3beta-hydroxyl-5-Delta steroids with anhydrous ferric chloride

Treatment of 3beta-hydroxyl-5-Delta steroids with anhydrous FeCl(3) in CH(2)Cl(2) afforded reasonable yields of the corresponding alkyl chlorides with a retention of configurations. The structures of the chlorine-exchanging products were determined by NMR and HRMS spectra. The absolute configurations were confirmed by X-ray crystal analysis of 3beta-chloro-androst-5-en-17-one. The generality and scope of the reaction were also investigated.     

The crystal and molecular structures of lithium trimethylenediaminetetraacetatoferrate(III) trihydrate Li[Fe(trdta)]·3H2O and sodium ethylenediamine-N,N′-diacetato-N,N′-di-3-propionatoferrate(II) pentahydrate Na[Fe(eddda)]·5H2O

The crystal structures of Li[Fe(trtda)]·3H₂O and Na[Fe(eddda)]·5H₂O (trtda = trimethylenediaminetetraacetate and eddda = ethylenediamine-N,N′-diacetate-N,N′-di-3-propionate) have been determined by single crystal X-ray diffraction techniques. The former crystal was monoclinic with the space group P2₁/n,a = 17.775(3),b = 10.261(1),c = 8.883(2)Å, β = 95.86(4)° and Z = 4. The latter was also monoclinic with the space group P2₁/n,a = 6.894(2),b = 20.710(6),c = 13.966(3)Å, β = 101.44(2)° and Z = 4. Both complex anions were found to adopt an octahedral six-coordinated structure with all of six ligand atoms of trdta⁴⁻ or eddda⁴⁻ coordinated to the Fe(III) ion, unlike the corresponding edta⁴⁻ complex which is usually seven-coordinate with the seventh coordination site occupied by H₂O. Of the three geometrical isomers possible for the eddda complex, the trans(O₅) isomer was actually found in the latter crystal. Factors determining the structural types of metal–edta complexes are discussed in detail.     

The crystal structures of oxidized forms of human peroxiredoxin 5 with an intramolecular disulfide bond confirm the proposed enzymatic mechanism for atypical 2-Cys peroxiredoxins

Peroxiredoxin 5 (PRDX5) belongs to the PRDX superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. PRDX5 is classified in the atypical 2-Cys subfamily of PRDXs. In this subfamily, the oxidized form of the enzyme is characterized by the presence of an intramolecular disulfide bridge between the peroxidatic and the resolving cysteine residues. We report here three crystal forms in which this intramolecular disulfide bond is indeed observed. The structures are characterized by the expected local unfolding of the peroxidatic loop, but also by the unfolding of the resolving loop. A new type of interface between PRDX molecules is described. The three crystal forms were not oxidized in the same way and the influence of the oxidizing conditions is discussed.     

A nanobeads amplified QCM immunosensor for the detection of avian influenza virus H5N1

As a potential pandemic threat to human health, there has been an urgent need for rapid detection of the highly pathogenic avian influenza (AI) H5N1 virus. In this study, magnetic nanobeads amplification based quartz crystal microbalance (QCM) immunosensor was developed as a new method and application for AI H5N1 virus detection. Polyclonal antibodies against AI H5N1 virus surface antigen HA (Hemagglutinin) were immobilized on the gold surface of the QCM crystal through self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA). Target H5N1 viruses were then captured by the immobilized antibodies, resulting in a change in the frequency. Magnetic nanobeads (diameter, 30nm) coated with anti-H5 antibodies were used for further amplification of the binding reaction between antibody and antigen (virus). Both bindings of target H5N1 viruses and magnetic nanobeads onto the crystal surface were further confirmed by environmental scanning electron microscopy (ESEM). The QCM immunosensor could detect the H5N1 virus at a titer higher than 0.0128 HA unit within 2h. The nanobeads amplification resulted in much better detection signal for target virus with lower titers. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent, and a linear correlation between the logarithmic number of H5N1 virus titers and frequency shift was found from 0.128 to 12.8 HA unit. No significant interference was observed from non-target subtypes such as AI subtypes H3N2, H2N2, and H4N8. The immunosensor was evaluated using chicken tracheal swab samples. This research demonstrated that the magnetic nanobeads amplification based QCM immunosensor has a great potential to be an alternative method for rapid, sensitive, and specific detection of AI virus H5N1 in agricultural, food, environmental and clinical samples.     

The crystal structure at 1.5 angstroms resolution of an RNA octamer duplex containing tandem G.U basepairs

The crystal structure of the RNA octamer, 5'-GGCGUGCC-3' has been determined from x-ray diffraction data to 1.5 angstroms resolution. In the crystal, this oligonucleotide forms five self-complementary double-helices in the asymmetric unit. Tandem 5'GU/3'UG basepairs comprise an internal loop in the middle of each duplex. The NMR structure of this octameric RNA sequence is also known, allowing comparison of the variation among the five crystallographic duplexes and the solution structure. The G.U pairs in the five duplexes of the crystal form two direct hydrogen bonds and are stabilized by water molecules that bridge between the base of guanine (N2) and the sugar (O2') of uracil. This contrasts with the NMR structure in which only one direct hydrogen bond is observed for the G.U pairs. The reduced stability of the r(CGUG)2 motif relative to the r(GGUC)2 motif may be explained by the lack of stacking of the uracil bases between the Watson-Crick and G.U pairs as observed in the crystal structure.     

Pyrazole and pyrimidine phenylacylsulfonamides as dual Bcl-2/Bcl-xL antagonists

5-Butyl-1,4-diphenyl pyrazole and 2-amino-5-chloro pyrimidine acylsulfonamides were developed as potent dual antagonists of Bcl-2 and Bcl-xL. Compounds were optimized for binding to the I88, L92, I95, and F99 pockets normally occupied by pro-apoptotic protein Bim. An X-ray crystal structure confirmed the proposed binding mode. Observation of cytochrome c release from isolated mitochondria in MV-411 cells provides further evidence of target inhibition. Compounds demonstrated submicromolar antiproliferative activity in Bcl-2/Bcl-xL dependent cell lines.     

Crystal structure of the 2'-specific and double-stranded RNA-activated interferon-induced antiviral protein 2'-5'-oligoadenylate synthetase

2'-5'-oligoadenylate synthetases are interferon-induced, double-stranded RNA-activated antiviral enzymes which are the only proteins known to catalyze 2'-specific nucleotidyl transfer. This crystal structure of a 2'-5'-oligoadenylate synthetase reveals a structural conservation with the 3'-specific poly(A) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2'- and 3'-specific nucleotidyl transferases. Comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. The 2'-5'-oligoadenylate synthetases are activated by viral double-stranded RNA in infected cells and initiate a cellular response by synthesizing 2'-5'-oligoadenylates, which in turn activate RNase L. This crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsRNA activation site that is probed by mutagenesis, thus providing structural insight into cellular recognition of viral double-stranded RNA.     

Characterization of eight Bacillus thuringiensis isolates originated from fecal samples of Fuzhou Zoo and Fuzhou Panda Center

In eight fecal samples of 5 herbivorous animals from Fuzhou Zoo and Fuzhou Panda Center were found eight Bacillus thuringiensis isolates. Obtained isolates were characterized by crystal microscopy, cry identification, and assay against the development of Eimeria tenella oocyst in chicken embryo. Bt WCB1, WCB2, WCB3 and WCB8, originated from horse, spotted deer, giant panda and lesser panda, respectively, all exhibited bipyramidal and cuboidal crystal and harbored the same cry genes, which were cry1Cb, cry1Db, cry1Fa, cry1Ib and cry2Ab. WCB7, a Bt isolate with bipyramidal crystal from zebra, showed a different cry pattern, cry1Cb, cry1Db, cry1Fa and cry1Ia. Bt WCB6, the third Bt isolate from giant panda, also contained bipyramidal crystal, however, harbored cry1Ib only. It was noted that Bt isolate WCB5 contained cuboidal crystal and harbored an unknown cry3 gene. It was very interesting that no cry genes were detected from Bt isolate WCB4 with dot-like crystal. Parasporal crystal proteins of WCB3, WCB4 and WCB7 obviously inhibited the development of E. tenella oocyst in chicken embryo.     

Efficient synthesis, spectral analysis and antimicrobial studies of nitrogen and sulfur containing spiro heterocycles from 2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-ones

Acetyl and propionyl group substituted thiadiazole derivatives (4a-4h, 5a-5h, 6a, 6b, 7a and 7b) have been synthesized by the cyclization of 2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-one thiosemicarbazones (2a-2h, 3a and 3b) with acetic anhydride/propionic anhydride and were characterized by Elemental analysis, IR, (1)H NMR and (13)C NMR spectral analysis. Single crystal X-ray diffraction has also been recorded for compounds 4c and 5a. From the NMR and Single crystal X-ray diffraction analysis, compounds 4b-4d, 4f-4h, 5b, 5c, 5f-5h, 6a, 7a and 7b were found to adopt twin-chair conformations whereas compounds 4a, 4e, 5a, 5d, 5e and 6b adopt chair and boat conformation of cyclohexane and piperidine rings, respectively. Besides, the synthesized compounds were screened for antibacterial and antifungal activities using serial dilution method. The microbiological analysis showed that the electron withdrawing function substituted phenyl group at C-2 and C-4 of azabicyclononane based thiadiazoles 4c/4h and 5c/5h exposed significant antimicrobial activity against Salmonella typhi, Escherichia coli, Klebsiella pneumoniae, Aspergillus flavus, Aspergillus niger and Candida albicans at MIC of 6.25 μg/ml.     

Synthesis, characterization and the first crystal structure of the Zn(II) complex of 4,6-O-ethylidine-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine.

4,6-O-Ethylidine-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine (H(3)L(1)) and N-(5-bromo-2-hydroxybenzylidene-4,6-O-ethylidine-beta-D-glucopyranosylamine (H(3)L(2)) molecules possessing a single bond C-1 single bond N double bond C(H) single bond moiety for metal-ion binding were synthesized by condensing the 4,6-O-ethylidene-beta-D-glucopyranosylamine with salicylaldehyde or 5-bromosalicylaldehyde. Complexes of these ligands with Zn(II) were isolated and characterized using elemental analysis, FTIR, UV-Vis absorption, NMR spectroscopic and FAB mass spectrometric techniques. The structure of the Zn(II) complex derived from H(3)L(1) was established for the first time by a single-crystal X-ray diffraction study. The anomeric nature of the saccharide moiety was established based on (1)H NMR studies and was confirmed by the crystal structure. Further, the structure and binding aspects of the ligand, and the coordination features of this in its Zn(II) complex were derived from the corresponding crystal structure.     

In vitro uptake of 140 kDa Bacillus thuringiensis nematicidal crystal proteins by the second stage juvenile of Meloidogyne hapla

Plant-parasitic nematodes (PPNs) are piercing/sucking pests, which cause severe damage to crops worldwide, and are difficult to control. The cyst and root-knot nematodes (RKN) are sedentary endoparasites that develop specialized multinucleate feeding structures from the plant cells called syncytia or giant cells respectively. Within these structures the nematodes produce feeding tubes, which act as molecular sieves with exclusion limits. For example, Heterodera schachtii is reportedly unable to ingest proteins larger than 28 kDa. However, it is unknown yet what is the molecular exclusion limit of the Meloidogyne hapla. Several types of Bacillus thuringiensis crystal proteins showed toxicity to M. hapla. To monitor the entry pathway of crystal proteins into M. hapla, second-stage juveniles (J2) were treated with NHS-rhodamine labeled nematicidal crystal proteins (Cry55Aa, Cry6Aa, and Cry5Ba). Confocal microscopic observation showed that these crystal proteins were initially detected in the stylet and esophageal lumen, and subsequently in the gut. Western blot analysis revealed that these crystal proteins were modified to different molecular sizes after being ingested. The uptake efficiency of the crystal proteins by the M. hapla J2 decreased with increasing of protein molecular mass, based on enzyme-linked immunosorbent assay analysis. Our discovery revealed 140 kDa nematicidal crystal proteins entered M. hapla J2 via the stylet, and it has important implications in designing a transgenic resistance approach to control RKN.     

1,3-Dioxane-4,6-dione-5-carboxamide-based inhibitors of human group IIA phospholipase A: X-ray structure of the complex and interfacial selection of inhibitors from a structural library.

A library of 109 1,3-dioxane-4,6-dione-5-carboxamides was prepared by solution-phase methods as potential inhibitors of human group IIa phospholipase A2. Tight binding inhibitors were found by an interfacial affinity selection method. The crystal structure of the secreted phospholipase A2 containing one of the inhibitors was determined, and it reveals the inhibitor-calcium bidendate coordination.     

Altered crystal violet permeability and lytic behavior in antibiotic-resistant and -sensitive mutants of Neisseria gonorrhoeae.

Wild-type, antibiotic-resistant and hypersensitive isogenic strains of Neisseria gonorrhoeae were studied for uptake of crystal violet, rates of autolysis, and response to lysozyme. Total uptake of crystal violet was similar in all strains at 0 C but varied significantly at 37 C. Mutation at the nonspecific resistance locus ery resulted in relative impermeability to crystal violet at 37 C, as compared to wild type. The penetration barrier to crystal violet at 37 C was overcome by addition of 5 mM ethylenediaminetetraacetic acid. Mutation at ery also resulted in reduced rates of autolysis and reduced sensitivity to high concentrations of lysozyme under conditions of divalent cation (Mg2+) depletion. In contrast, mutation at the nonspecific drug hypersensitivity locus env resulted in increased uptake of crystal violet at 37 C, due to increased binding of dye to crude envelope as well as increased penetration into cytoplasm. The env mutants were also more rapidly autolytic and more sensitive to lysozyme than wild type in the absence of Mg2+. These results suggest that the cell envelopes of ery mutants are more stable and less permeable and those of env mutants are less stable and more permeable than wild-type strains.     

Selenium derivatization of nucleic acids for crystallography

The high-resolution structure of the DNA (5′-GTGTACA-C-3′) with the selenium derivatization at the 2′-position of T2 was determined via MAD and SAD phasing. The selenium-derivatized structure (1.28 Å resolution) with the 2′-Se modification in the minor groove is isomorphorous to the native structure (2.0 Å). To directly compare with the conventional bromine derivatization, we incorporated bromine into the 5-postion of T4, determined the bromine-derivatized DNA structure at 1.5 Å resolution, and found that the local backbone torsion angles and solvent hydration patterns were altered in the structure with the Br incorporation in the major groove. Furthermore, while the native and Br-derivatized DNAs needed over a week to form reasonable-size crystals, we observed that the Se-derivatized DNAs grew crystals overnight with high-diffraction quality, suggesting that the Se derivatization facilitated the crystal formation. In addition, the Se-derivatized DNA sequences crystallized under a broader range of buffer conditions, and generally had a faster crystal growth rate. Our experimental results indicate that the selenium derivatization of DNAs may facilitate the determination of nucleic acid X-ray crystal structures in phasing and high-quality crystal growth. In addition, our results suggest that the Se derivatization can be an alternative to the conventional Br derivatization.     

Intermolecular interactions in the crystal structures of potential HIV-1 integrase inhibitors

2-[(2,5-dichloro-4-nitro-phenylamino)-methoxy-methyl]-8-hydroxy-quinoline 1 and 2-methyl-quinoline-5,8-dione-5-oxime 2 were obtained as potential HIV-1 integrase inhibitors and analyzed by X-ray crystallography. Semiempirical theoretical calculations of energy preferred conformations were also carried out. The crystal structures of both compounds are stabilized via hydrogen bonds and pi-pi stacking interactions. The planarity of compound 1 is caused by intramolecular hydrogen bonds.     

Progress in cytochrome P450 active site modeling

Models capable of predicting the possible involvement of cytochromes P450 in the metabolism of drugs or drug candidates are important tools in drug discovery and development. Ideally, functional information would be obtained from crystal structures of all the cytochromes P450 of interest. Initially, only crystal structures of distantly related bacterial cytochromes P450 were available-comparative modeling techniques were used to bridge the gap and produce structural models of human cytochromes P450, and thereby obtain some useful functional information. A significant step forward in the reliability of these models came four years ago with the first crystal structure of a mammalian cytochrome P450, rabbit CYP2C5, followed by the structures of two human enzymes, CYP2C8 and CYP2C9, and a second rabbit enzyme, CYP2B4. The evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism, is presented as a case study.