Interactions of aromatic carboxylic acids with 8-aminoquinoline: Synthesis and the crystal structures of the proton-transfer compounds of 8-aminoquinoline with nitro-substituted benzoic acids

Proton-transfer compounds of 8-aminoquinoline with the nitro-substituted aromatic carboxylic acids 3-nitrobenzoic acid, [(C9H9N2+)(C7 H4NO4-)] (1), 4-nitrobenzoic acid, [(C9H9N2+) (C7H5NO4)] (2), 3,5-dinitrobenzoic acid, [(C9H9N2+)(C7 H3N2O6-)] (3), 5-nitrosalicylic acid, [(C9H9N2+) (C7H4NO5-)] (4) and 3,5-dinitrosalicylic acid, [(C9H9N2+) (C7H3N2O7-)] (5) have been prepared and characterized by using both infrared spectroscopy and single-crystal X-ray diffraction methods [(1) (4) and (5)]. In all compounds, protonation of the quinoline nitrogen occurs together with primary hydrogen-bonding interactions involving this group and the carboxylate group of the acid, while further peripheral associations result predominantly in simple chain polymeric structures. The attempted preparation of the adduct with 2,4,6-trinitrobenzoic acid gave the unstable, neutral, essentially 1:1 adduct with the decarboxylation product 1,3,5-trinitrobenzene, the X-ray crystal structure of which indicates the stoichiometry [(C9H8N2)0.6(C6 H3N3O6)0.8] (6)

5-Nitrosalicylic acid and its proton-transfer compounds with aliphatic Lewis bases

The crystal structures of the proton-transfer compounds of 5-nitrosalicylic acid (5-nsa) with morpholine (morph), hexamethylenetetraminc (hmt), and ethylenediamine (en) have been determined and their solid-state packing structures described. The compounds are [(morph)+(5-nsa) -] 1, [(hmt)+(5-nsa)-·H2O] 2, and [(en)2+(5-nsa)-·H2O] 3. In all compounds, protonation of the hetero-nitrogen of the Lewis base occurs. With 1, the 5-nsa anions and the morpholine cations lie, respectively, in or across crystallographic mirror planes and are linked within the planes by hydrogen-bonding interactions through the aminium group and the carboxylic and phenolic oxygens of the anionic 5-nsa species giving a two-dimensional sheet polymer. Compound 2 is an unusual structure with the planar 5-nsa anions lying within pseudo minor planes and cyclically linked by duplex water bridges through a single carboxylate oxygen into centrosymmetric dimers. The hmt cation molecules are disordered across the pseudo mirror and are strongly linked by N+-H ... O hydrogen bonds only to the water molecules with peripheral weak hmt C-H ... O hydrogen bonds extending the dimer within and between the dimer planes. Compound 3 is a network polymer comprised of the 5-nsa anions, the en dianions, and the water molecule in an extensive hydrogen-bonded structure

A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution

Crystals of the RNA-dependent RNA polymerase catalytic domain from the dengue virus NS5 protein have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration. These crystals diffract to 1.85 Å resolution and are thus suitable for a structure-based drug-design program

A scintillation proximity assay for dengue virus NS5 2'-O-methyltransferase-kinetic and inhibition analyses.

Dengue virus (DENV) NS5 possesses methyltransferase (MTase) activity at its N-terminal amino acid sequence and is responsible for formation of a type 1 cap structure, m(7)GpppAm(2'-O) in the viral genomic RNA. Optimal in vitro conditions for DENV2 2'-O-MTase activity were characterized using purified recombinant protein and a short biotinylated GTP-capped RNA template. Steady-state kinetics parameters derived from initial velocities were used to establish a robust scintillation proximity assay for compound testing. Pre-incubation studies showed that MTase-AdoMet and MTase-RNA complexes were equally catalytically competent and the enzyme supports a random bi bi kinetic mechanism. The assay was validated with competitive inhibitory agents, S-adenosyl-homocysteine and two homologues, sinefungin and dehydrosinefungin. A GTP-binding pocket present at the N-terminal of DENV2 MTase was previously postulated to be the cap-binding site. Interestingly, inhibition of the enzyme by GTP was two-fold lower than with RNA cap analogues, G[5']ppp[5']A and m(7)G[5']ppp[5']A and about three-fold poorer than a two-way methylated analogue, m(7)G[5']ppp[5']m(7)G. This assay allows rapid and highly sensitive detection of 2'-O-MTase activity and can be readily adapted for high-throughput screening for inhibitory compounds. It is suitable for determination of enzymatic activities of a wide variety of RNA capping MTases

Expression, purification, crystallization, and preliminary X-ray analysis of the N-terminal domain of Escherichia coli adenylyl transferase

A soluble N-terminal domain of the Escherichia coli adenylyl transferase (ATase) is responsible for deadenylylation activity of the intact enzyme. Previous studies of the deadenylylation activity have involved a fragment, AT-N423 (residues 1 to 423), which was extended by 17 amino acids to give AT-N440. This new domain is truncated at the end of a predicted helix and prior to a Q-linker. The domain was found to be very soluble and stable so that it could be purified to homogeneity and crystallized. This construct has deadenylylation activity that is independent of the low nitrogen status indicator PII-UMP. The crystals belong to space group P3121 or its enantiomorph P3221 with a=b=116.6 Å and c=67.6 Å

Expression, purification, crystallization, and preliminary X-ray analysis of the N-terminal domain of Escherichia coli adenylyl transferase

A soluble N-terminal domain of the Escherichia coli adenylyl transferase (ATase) is responsible for deadenylylation activity of the intact enzyme. Previous studies of the deadenylylation activity have involved a fragment, AT-N423 (residues 1 to 423), which was extended by 17 amino acids to give AT-N440. This new domain is truncated at the end of a predicted helix and prior to a Q-linker. The domain was found to be very soluble and stable so that it could be purified to homogeneity and crystallized. This construct has deadenylylation activity that is independent of the low nitrogen status indicator PII-UMP. The crystals belong to space group P3121 or its enantiomorph P3221 with a = b = 116.6 Å and c = 67.6 Å

Expression, purification and crystallization of the C-­terminal domain of Escherichia coli adenylyltransferase

The C-terminal domain of adenylyltransferase (ATase) from Escherichia coli has been overexpressed, purified and crystallized in a form suitable for structure analysis

The Escherichia coli signal transducers PII (GlnB) and GlnK form heterotrimers in vivo: Fine tuning the nitrogen signal cascade

The PII protein is Escherichia coli's cognate transducer of the nitrogen signal to the NRII (NtrB)/NRI (NtrC) two-component system and to adenylyltransferase. Through these two routes, PII regulates both amount and activity of glutamine synthetase. GlnK is the recently discovered paralogue of PII, with a similar trimeric x-ray structure. Here we show that PII and GlnK form heterotrimers, in E. coli grown in nitrogen-poor medium. In vitro, fully uridylylated heterotrimers of the two proteins stimulated the deadenylylation activity of adenylyltransferase, albeit to a lower extent than homotrimeric PII-UMP. Fully uridylylated GlnK did not stimulate, or hardly stimulated, the deadenylylation activity. We propose that uridylylated PII/GlnK heterotrimers fine-regulate the activation of glutamine synthetase. The PII/GlnK couple is a first example of prokaryotic signal transducer that can form heterotrimers. Advantages of hetero-oligomer formation as molecular mechanism for fine-regulation of signal transduction are discussed

Patterning of the vertebrate ventral spinal cord

We review investigations that have lead to a model of how the ventral spinal cord of higher vertebrate embryos is patterned during development. Central to this model is the secreted morphogen protein, Sonic hedgehog. There is now considerable evidence that this molecule acts in a concentration-dependent manner to direct the development of the spinal cord. Recent studies have suggested that two classes of homeodomain proteins are induced by threshold concentrations of Sonic hedgehog. Reciprocal inhibition between the two classes acts to convert the continuous gradient of Sonic hedgehog into defined domains of transcription factor expression. However, a number of aspects of ventral spinal cord patterning remain to be elucidated. Some issues currently under investigation involve temporal aspects of Shh-signalling, the role of other signals in ventral patterning and the characterisation of ventral interneurons. In this review, we discuss the current state of knowledge of these issues and present some preliminary studies aimed at furthering understanding of these processes in spinal cord patterning