Molecular details of quinolone–DNA interactions: solution structure of an unusually stable DNA duplex with covalently linked nalidixic acid residues and non-covalent complexes derived from it

Quinolones are antibacterial drugs that are thought to bind preferentially to disturbed regions of DNA. They do not fall into the classical categories of intercalators, groove binders or electrostatic binders to the backbone. We solved the 3D structure of the DNA duplex (ACGCGU-NA)(2), where NA denotes a nalidixic acid residue covalently linked to the 2′-position of 2′-amino-2′-deoxyuridine, by NMR and restrained torsion angle molecular dynamics (MD). In the complex, the quinolones stack on G:C base pairs of the core tetramer and disrupt the terminal A:U base pair. The displaced dA residues can stack on the quinolones, while the uracil rings bind in the minor groove. The duplex-bridging interactions of the drugs and the contacts of the displaced nucleotides explain the high UV-melting temperature for d(ACGCGU-NA)(2) of up to 53°C. Further, non-covalently linked complexes between quinolones and DNA of the sequence ACGCGT can be generated via MD using constraints obtained for d(ACGCGU-NA)(2). This is demonstrated for unconjugated nalidixic acid and its 6-fluoro derivative. The well-ordered and tightly packed structures thus obtained are compatible with a published model for the quinolone–DNA complex in the active site of gyrases

Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins

The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where ‘n’ can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5′ of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel α-helical ‘tweezer’-like structure

Asymmetric DNA recognition by the OkrAI endonuclease, an isoschizomer of BamHI

Restriction enzymes share little or no sequence homology with the exception of isoschizomers, or enzymes that recognize and cleave the same DNA sequence. We present here the structure of a BamHI isoschizomer, OkrAI, bound to the same DNA sequence (TATGGATCCATA) as that cocrystallized with BamHI. We show that OkrAI is a more minimal version of BamHI, lacking not only the N- and C-terminal helices but also an internal 310 helix and containing β-strands that are shorter than those in BamHI. Despite these structural differences, OkrAI recognizes the DNA in a remarkably similar manner to BamHI, including asymmetric contacts via C-terminal ‘arms’ that appear to ‘compete’ for the minor groove. However, the arms are shorter than in BamHI. We observe similar DNA-binding affinities between OkrAI and BamHI but OkrAI has higher star activity (at 37°C) compared to BamHI. Together, the OkrAI and BamHI structures offer a rare opportunity to compare two restriction enzymes that work on exactly the same DNA substrate

Light meson spectroscopy from Dalitz plot analyses ηc decays to η0K + K − , η0π + π − , and ηπ + π − produced in two-photon interactions

We study the processes γγ → ηc → η0KþK−, η0πþπ−, and ηπþπ− using a data sample of 519 fb−1 recorded with the BABAR detector operating at the SLAC PEP-II asymmetric-energy e+e− collider at center-of-mass energies at and near the Υ(nS) (n = 2, 3, 4) resonances. This is the first observation of the decay ηc → η0KþK− and we measure the branching fraction Γðηc → η0KþK−Þ=ðΓðηc → η0πþπ−Þ 1⁄4 0.644 0.039stat 0.032sys. Significant interference is observed between γγ → ηc → ηπþπ− and the nonresonant two-photon process γγ → ηπþπ−. A Dalitz plot analysis is performed of ηc decays to η0KþK−, η0πþπ−, and ηπþπ−. Combined with our previous analysis of ηc → KK ̄ π, we measure the K 0ð1430Þ parameters and the ratio between its η0K and πK couplings. The decay ηc → η0πþπ− is dominated by the f0ð2100Þ resonance, also observed in J=ψ radiative decays. A new a0(1700)→ ηπ resonance is observed in the ηc → ηπþπ− channel. We also compare ηc decays to η and η0 final states in association with scalar mesons as they relate to the identification of the scalar glueball

Middle East - North Africa and the millennium development goals : implications for German development cooperation

          Closed-loop controlled combustion is a promising technique to improve the overall performance of internal combustion engines and Diesel engines in particular. In order for this technique to be implemented some form of feedback from the combustion process is required. The feedback signal is processed and from it combustionrelated parameters are computed. These parameters are then fed to a control process which drives a series of outputs (e.g. injection timing in Diesel engines) to control their values. This paper’s focus lies on the processing and computation that is needed on the feedback signal before this is ready to be fed to the control process as well as on the electronics necessary to support it. A number of feedback alternatives are briefly discussed and for one of them, the in-cylinder pressure sensor, the CA50 (crank angle in which the integrated heat release curve reaches its 50% value) and the IMEP (Indicated Mean Effective Pressure) are identified as two potential control variables. The hardware architecture of a system capable of calculating both of them on-line is proposed and necessary feasibility size and speed considerations are made by implementing critical blocks in VHDL targeting a flash-based Actel ProASIC3 automotive-grade FPGA

Semiautomated model building for RNA crystallography using a directed rotameric approach

Structured RNA molecules play essential roles in a variety of cellular processes; however, crystallographic studies of such RNA molecules present a large number of challenges. One notable complication arises from the low resolutions typical of RNA crystallography, which results in electron density maps that are imprecise and difficult to interpret. This problem is exacerbated by the lack of computational tools for RNA modeling, as many of the techniques commonly used in protein crystallography have no equivalents for RNA structure. This leads to difficulty and errors in the model building process, particularly in modeling of the RNA backbone, which is highly error prone due to the large number of variable torsion angles per nucleotide. To address this, we have developed a method for accurately building the RNA backbone into maps of intermediate or low resolution. This method is semiautomated, as it requires a crystallographer to first locate phosphates and bases in the electron density map. After this initial trace of the molecule, however, an accurate backbone structure can be built without further user intervention. To accomplish this, backbone conformers are first predicted using RNA pseudotorsions and the base-phosphate perpendicular distance. Detailed backbone coordinates are then calculated to conform both to the predicted conformer and to the previously located phosphates and bases. This technique is shown to produce accurate backbone structure even when starting from imprecise phosphate and base coordinates. A program implementing this methodology is currently available, and a plugin for the Coot model building program is under development

Structural study of TcaR and its complexes with multiple antibiotics from Staphylococcus epidermidis

TcaR and IcaR are a weak and a strong negative regulator of transcription of the ica locus, respectively, and their presence prevents the poly-N-acetylglucosamine production and biofilm formation in Staphylococcus epidermidis. Although TcaR was shown to interact with the ica promoter, the precise binding region and the mechanism of interaction remained unclear. Here we present the 3D structure of TcaR in its apo form and in complex with salicylate as well as several aminoglycoside and β-lactam antibiotics. A comparison of the native and complex TcaR structures indicates that the mechanism of regulation involves a large conformational change in the DNA-binding lobe. Here, we deduced the consensus binding sequence of two [∼TTNNAA] hexamers embedded in a 16 bp sequence for a TcaR dimer. Six TcaR dimers bind specifically to three approximately 33 bp segments close to the IcaR binding region with varying affinities, and their repressor activity is directly interfered by salicylate and different classes of natural antimicrobial compounds. We also found in this study that the antimicrobial compounds we tested were shown not only to inhibit TcaR–DNA interaction but also to further induce biofilm formation in S. epidermidis in our in vivo assay. The results support a general mechanism for antibiotics in regulating TcaR–DNA interaction and thereby help understand the effect of antibiotic exposure on bacterial antibiotic resistance through biofilm formation

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

B12-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme