Identification of Specific DNA Binding Residues in the TCP Family of Transcription Factors in Arabidopsis

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an similar to 60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix ( bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors.

Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the D-Ala-D-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetyl-glucosamine and N-acetyl-muramic acid-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. beta-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a beta-lactamase and is not trapped as an acyl intermediate with beta-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.

The slowing down of galaxy disks in dissipationless minor mergers

We have investigated the impact of dissipationless minor galaxy mergers on the angular momentum of the remnant. Our simulations cover a range of initial orbital characteristics, and the system consists of a massive galaxy with a bulge and disk merging with a much less massive (one-tenth or one-twentieth) gasless companion that has a variety of morphologies (disk-or elliptical-like) and central baryonic mass concentrations. During the process of merging, the orbital angular momentum is redistributed into the internal angular momentum of the final system; the internal angular momentum of the primary galaxy can increase or decrease depending on the relative orientation of the orbital spin vectors (direct or retrograde), while the initially nonrotating dark matter halo always gains angular momentum. The specific angular momentum of the stellar component always decreases independently of the orbital parameters or morphology of the satellite, the decrease in the rotation velocity of the primary galaxy is accompanied by a change in the anisotropy of the orbits, and the ratio of rotation speed to velocity dispersion of the merger remnant is lower than the initial value, not only because of an increase in the dispersion but also of the slowing-down of the disk rotation. We briefly discuss several astrophysical implications of these results, suggesting that minor mergers do not cause a "random walk" process of the angular momentum of the stellar disk component of galaxies, but rather a steady decrease. Minor mergers may play a role in producing the large scatter observed in the Tully-Fisher relation for S0 galaxies, as well as in the increase of the velocity dispersion and the decrease in upsilon/sigma at large radii as observed in S0 galaxies.

Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein

A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination.

Differential binding of peanut agglutinin with lipopolysaccharide of homologous and heterologous Rhizobium

To establish the crucial role of lipopolysaccharide in the initial recognition event of symbiotic peanut-Rhizobium system the ability of various surface polysaccharides isolated from Bradyrhizobium arachis to inhibit the precipitin reaction between peanut agglutinin and asialoganglioside: deoxycholate (1:1) micelles was estimated. It was compared with that of nonsymbiotic systems e.g. Bradyrhizobium japonicum, Bradyrhizobium ciceris and Escherichia coli. Peanut agglutinin was found to interact more strongly with the lipopolysaccharide of Bradyrhizobium arachis than the exopolysaccharide or capsular polysaccharide. The inhibitory capacity of lipopolysaccharides from homologous and heterologous Bradyrhizobium as measured in terms of the concentration necessary for 50 percent inhibition of precipitin reaction were 1428, 500, 410, and 277 times less than that of lactose for Bradyrhizobium arachis, B. japonicum, B. ciceris and Escherichia coli, respectively. These results support that host lectin peanut agglutinin can recognize homologous Bradyrhizobium lipopolysaccharide by virtue of its binding specificity of higher magnitude.

Solution cation-binding properties of segmented polyethylene oxide-A C-13 NMR spectroscopic study

Two segmented polyethylene oxides, SPEO-3 and SPEO-4, were prepared using a novel transetherification methodology. Their structures were confirmed by H-1 and C-13 NMR spectroscopy. The complexation of these SPEO's with alkali-metal ions in solution was investigated by C-13 NMR spectroscopy. The mole-fraction method was used to determine the complexation ratio of SPEO with LIClO4 at 25 degrees C, which showed that these formed 1:1 (polymer repeat unit/salt) complexes. The association constant, K, for the complex formation was calculated from the variation of the chemical shift values with salt concentration, using a standard nonlinear least-square fitting procedure. The maximum change in chemical shift (Delta delta) and the K values suggest that both SPEO-3 and SPEO-4 formed stronger complexes with lithium salts than with sodium salts. Unexpectedly, the K values were found to be different, when the variation of delta of different carbons was used in the fitting procedure. This suggests that several possible complexed species may be in equilibrium with the uncomplexed one. Structurally similar model compounds were also prepared and their complexation studies indicated that all of them also formed 1:1 complexes with Li salts. Interestingly, it was observed that the polymers gave higher K values suggesting the formation of more stable complexes in polymers when compared to the model analogues. (C) 2000 John Wiley & Sons, Inc.

Computer modelling approach to study the modes of binding of alpha- and beta-anomers of D-galactose, D-fucose and D-glucose to L-arabinose-binding protein

The modes of binding of alpha- and beta-anomers of D-galactose, D-fucose and D-glucose to L-arabinose-binding protein (ABP) have been studied by energy minimization using the low resolution (2.4 A) X-ray data of the protein. These studies suggest that these sugars preferentially bind in the alpha-form to ABP, unlike L-arabinose where both alpha- and beta-anomers bind almost equally. The best modes of binding of alpha- and beta-anomers of D-galactose and D-fucose differ slightly in the nature of the possible hydrogen bonds with the protein. The residues Arg 151 and Asn 232 of ABP from bidentate hydrogen bonds with both L-arabinose and D-galactose, but not with D-fucose or D-glucose. However in the case of L-arabinose, Arg 151 forms hydrogen bonds with the hydroxyl group at the C-4 atom and the ring oxygen, whereas in case of D-galactose it forms bonds with the hydroxyl groups at the C-4 and C-6 atoms of the pyranose ring. The calculated conformational energies also predict that D-galactose is a better inhibitor than D-fucose and D-glucose, in agreement with kinetic studies. The weak inhibitor D-glucose binds preferentially to one domain of ABP leading to the formation of a weaker complex. Thus these studies provide information about the most probable binding modes of these sugars and also provide a theoretical explanation for the observed differences in their binding affinities.

Plasticity in the primary binding site of galactose/N-acetylgalactosamine-specific lectins - Implication of the C-H center dot center dot center dot O hydrogen bond at the specificity-determining C-4 locus of the saccharide in 4-methoxygalactose recognition by jacalin and winged bean (basic) agglutinin I

It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of C-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The alpha-and beta-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C-H ... O-type hydrogen-bond(s) in protein-carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular.

Computer simulation of protein—carbohydrate complexes: application to arabinose-binding protein and pea lectin

The CCEM method (Contact Criteria and Energy Minimisation) has been developed and applied to study protein-carbohydrate interactions. The method uses available X-ray data even on the native protein at low resolution (above 2.4 Å) to generate realistic models of a variety of proteins with various ligands.The two examples discussed in this paper are arabinose-binding protein (ABP) and pea lectin. The X-ray crystal structure data reported on ABP-β-l-arabinose complex at 2.8, 2.4 and 1.7 Å resolution differ drastically in predicting the nature of the interactions between the protein and ligand. It is shown that, using the data at 2.4 Å resolution, the CCEM method generates complexes which are as good as the higher (1.7 Å) resolution data. The CCEM method predicts some of the important hydrogen bonds between the ligand and the protein which are missing in the interpretation of the X-ray data at 2.4 Å resolution. The theoretically predicted hydrogen bonds are in good agreement with those reported at 1.7 Å resolution. Pea lectin has been solved only in the native form at 3 Å resolution. Application of the CCEM method also enables us to generate complexes of pea lectin with methyl-α-d-glucopyranoside and methyl-2,3-dimethyl-α-d-glucopyranoside which explain well the available experimental data in solution.

Isolation and characterization of a gonadotropin receptor binding inhibitor from porcine follicular fluid

The presence of a gonadotropin receptor binding inhibitor in pooled porcine follicular fluid has been demonstrated. Porcine follicular fluid fractionation on DE-32 at near neutral pH, followed by a cation exchange chromatography on SPC-50 and Cibacron blue affinity chromatography, yielded a partially purified gonadotropin receptor binding inhibitor (GI-4). The partially purified GI binding inhibitor inhibited the binding of both 125I labelled hFSH and hCG to rat ovarian receptor preparation. SDS electrophoresis of radioiodinated partially purified GI followed by autoradiography made it possible to identify the binding component as a protein of molecular weight of 80000. Subjecting 125I labelled GI-4 to chromatography on Sephadex G-100 helped obtain a homogeneous material, Gl-5. The 125I labelled GI-5 exhibited in its binding to ovarian membrane preparations characteristics typical of a ligand-receptor interaction such as saturability, sensitivity to reaction conditions as time, ligand and receptor concentrations and finally displaceability by unlabelled inhibitor as well as FSH and hCG in a dose dependent manner. This material could bind ovarian receptors for both FSH and LH, its binding being inhibited by added FSH or hCG in a dose dependent manner.

Amido binding to ReO3+ core: Synthesis, structure and intermolecular interactions

The light green coloured complexes of general formula [(ReO)-O-V(L)CI(OH2)]Cl have been synthesised in good yields by reacting [RcvOCl(3)(AsPh3)21 with HL in dichloromethane in dinitrogen atmosphere. Here, L- is the deprotonated form of N',N'-bis(2-pyridylmethyl)amine (HL1); N-(2-pyridylmethyl)-N',N'-dimethylethylenediamine (HL2) and N-(2-pyridylmethyl)-N',N-diethylethylenediamine (HL3). Single crystal X-ray structure determination of [(ReO)-O-V(L-1)Cl(OH2)Cl confirms the amido binding of ReO3+ species. In the solid state of [(ReO)-O-V(L-1)Cl(OH2)]Cl, the coordinated and counter chloride ions are engaged in Re-Cl... H-C(ring), Cl...H-C(ring) and Re(OH2)...Cl hydrogen bonding and forming of a supramolecular network in the solid state. The subunit of the supramolecular network consists of one eight-membered and two nine-membered hydrogen bonded rings. The average diameters of eight-membered and nine-membered rings are similar to 3.70 and similar to 5.26 angstrom, respectively.

Ferrocene-Promoted Photoactivated DNA Cleavage and Anticancer Activity of Terpyridyl Copper(II) Phenanthroline Complexes

Ferrocene-appended copper(II) complexes [Cu( Fc-tpy)(B)](ClO4)(2) (1-3) and [Cu(Ph-tpy)(dppz)](ClO4)(2) (4) as control, where Fc-tpy is 4'-ferroceny1-2,2':6',2 ''-terpyridine, Ph-tpy is 4'-pheny1-2,2':6',2 ''-terpyridine, and B is a phenanthroline base, viz., 1,10-phenanthroline (phen, 1), dipyridoquinoxaline (dpq, 2), and dipyridophenazine (dppz, 3), were prepared and structurally characterized, and their DNA binding, photoactivated DNA cleavage activity, and cytotoxic properties were studied [Fe = (eta(5)-C5H4)Fe-11(eta(5)-C5H5)]. Complexes 1 and 3 as hexafluorophosphate salts were structurally characterized by X-ray crystallography. Molecular structures of [Cu(Fc-tpy)(phen)](PF6)(2) (1a) and [Cu(Fc-tpy)(dppz)](PF6)(2)center dot MeCN (3a center dot MeCN) show a distorted square-pyramidal geometry at copper(II), with the Fc-tpy ligand and the phenanthroline base showing respective tridentate and bidentate binding modes. The phenanthroline base exhibits axial-equatorial bonding, while the Fc-tpy ligand binds at the basal plane. The complexes showed quasi-reversible cyclic voltammetric responses near 0.45 and -0.3 V vs SCE in aqueous DMF-0.1 M KCl assignable to the Fc(+)-Fc and Cu(II) Cu(1) redox couples, respectively. The complexes bind to DNA, giving K-b values of 1.4 x 10(4) to 5.6 x 10(5) M-1 in the order 4 similar to 3 > 2 > 1. Thermal denaturation and viscometric titration data suggest groove and/or partial intercalative mode of DNA binding of the complexes. The complexes showed chemical nuclease activity in the presence of 3-mercaptopropionic acid (0.5 mM) or H2O2 (0.25 mM). Complexes 2-4 showed plasmid DNA cleavage activity in visible light, forming (OH)-O-center dot radicals. The Fc-tpy complex 3 showed better DNA photocleavage activity than its Ph-tpy analogue. The ferrocene moiety in the dppz complex 3 makes it more photocytotoxic than the Ph-tpy analogue 4 in HeLa cells.