Ligand-DNA interaction in a nanocage of reverse micelle

We have studied intercalation of ethidium bromide (EB) to genomic DNA encapsulated in a nanospace of an anionic AOT reverse micelle (RM). Circular dichroism (CD) study on the DNA in the RM reveals its condensed form. Here, we have used temporal decay-associated spectra (DAS) and time-resolved area normalized emission spectral (TRANES) techniques to investigate EB-binding to condensed DNA because the interference of emission from unbound EB in the RM makes conventional steady state and picosecond resolved fluorescence spectroscopic techniques challenging. The binding affinity of the ligand EB with the DNA in the RM is found to increase with the size of the RM, reflecting the effect of lessening of DNA condensation on the binding affinity. CD spectra of the DNA in the RM with various sizes indicate the structural change of the condensed DNA with reverse micellar size. DAS and TRANES techniques along with dynamic light scattering studies of the EB-DNA complex in the RM further reveal two kinds of binding modes of the ligand with the condensed DNA even in essentially monodispersed RMs. To investigate the role of RM on the ligand binding and secondary structure of the DNA, we have also studied complexation of EB with two synthetic self-complimentary oligonucleotides of sequences (CGCAAATTTGCG)2 and (CGCGCGCGCGCG)2 in the RM.     

The Mode of Binding of Carbohydrate in Ricin D

Dehydro-L-ascorbic acid (DAA) exists mainly in its C2 hydrated bicyclic form (5) in an aqueous solution, and monocyclic DAA (3), which is the expected reaction product immediately after the oxidation of AA, has not been observed by NMR spectroscopy. The formation mechanism for 5 from 3 and the stability of 5 were examined by the semi-empirical molecular orbital method (MOPAC). It was indicated that the protonation reaction was the key step in the formation of 5, therefore, the formation of 5 is thought to be more difficult under physiological conditions which mostly involve in the neutral or slightly alkaline state. However, by NMR, it was confirmed that, even in a neutral or slightly alkaline state very close to physiological conditions, the predominant form of DAA existing in an aqueous solution immediately after the enzymatic oxidation of AA was confirmed to be 5, although the possible existence of other forms of DAA at very low concentrations could not be completely excluded.     

Bayesian Analysis of Growth Curves Using Mixed Models Defined by Stochastic Differential Equations

Summary Growth curve data consist of repeated measurements of a continuous growth process over time in a population of individuals. These data are classically analyzed by nonlinear mixed models. However, the standard growth functions used in this context prescribe monotone increasing growth and can fail to model unexpected changes in growth rates. We propose to model these variations using stochastic differential equations (SDEs) that are deduced from the standard deterministic growth function by adding random variations to the growth dynamics. A Bayesian inference of the parameters of these SDE mixed models is developed. In the case when the SDE has an explicit solution, we describe an easily implemented Gibbs algorithm. When the conditional distribution of the diffusion process has no explicit form, we propose to approximate it using the Euler–Maruyama scheme. Finally, we suggest validating the SDE approach via criteria based on the predictive posterior distribution. We illustrate the efficiency of our method using the Gompertz function to model data on chicken growth, the modeling being improved by the SDE approach.     

Structure and Mode of Peptide Binding of Pheromone Receptor PrgZ

We present the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and OppA) explains the high specificity of PrgZ for hydrophobic heptapeptides versus the promiscuity of peptide binding in the homologous proteins.     

Different Binding Mode in AT and GC Sequences for Unfused-Aromatic Dications

Abstract

We have previously synthesized a 2,5-diphenylfuranamidine dication (4) and presented evidence that this compound binds to AT sequences in DNA by a minor-groove interaction mode but binds to GC sequences by intercalation (1,2). To probe these sequence-dependent binding modes in more detail, and particularly to obtain additional evidence for the binding mode in GC rich sequences, we have synthesized and studied the DNA complexes of 1–3 which have the furan ring of 4 replaced by 2,6-substituted pyridine (1), pyrimidine (2), or triazine (3) ring systems. The three compounds with a six-membered central ring system bind to AT DNA sequences more weakly than the furan compound, but retain the minor-groove binding mode. The pyridine and pyrimidine derivatives bind to GC sequences of DNA more strongly than the furan, but the triazine derivative binds more weakly. The aromatic proton signals of 1–3, as previously observed with 4 shift upfield by approximately 0.5 ppm or greater on complex formation with polyd(G-C)₂. This and other spectroscopic as well as viscosity and kinetics results indicate that 1–4 bind to GC sites in DNA by intercalation. A nonclassical intercalation model, with the twisted-unfused, aromatic ring system intercalated into an intercalation site of matching structure can explain all of our and the literature results for the GC binding mode of these unfused, aromatic compounds.     

Molecular Dynamics Reveal Binding Mode of Glutathionylspermidine by Trypanothione Synthetase

The trypanothione synthetase (TryS) catalyses the two-step biosynthesis of trypanothione from spermidine and glutathione and is an attractive new drug target for the development of trypanocidal and antileishmanial drugs, especially since the structural information of TryS from Leishmania major has become available. Unfortunately, the TryS structure was solved without any of the substrates and lacks loop regions that are mechanistically important. This contribution describes docking and molecular dynamics simulations that led to further insights into trypanothione biosynthesis and, in particular, explains the binding modes of substrates for the second catalytic step. The structural model essentially confirm previously proposed binding sites for glutathione, ATP and two Mg²⁺ ions, which appear identical for both catalytic steps. The analysis of an unsolved loop region near the proposed spermidine binding site revealed a new pocket that was demonstrated to bind glutathionylspermidine in an inverted orientation. For the second step of trypanothione synthesis glutathionylspermidine is bound in a way that preferentially allows N¹-glutathionylation of N⁸-glutathionylspermidine, classifying N⁸-glutathionylspermidine as the favoured substrate. By inhibitor docking, the binding site for N⁸-glutathionylspermidine was characterised as druggable.     

医院整体医疗管理模式探索历程与实施成效

医院整体医疗管理模式是以医学整体论、生物—心理—社会医学模式为指导,为满足社会民众的健康需求而创新的医院医疗管理新模式。经过10多年的实践与探索,拓宽了院前院后医疗保健功能,提升了医疗质量与医疗服务效能,融洽了医患关系,为医院赢得了良好的声誉,成效显著。     

Antigen Binding to Secretory Immunoglobulin A Results in Decreased Sensitivity to Intestinal Proteases and Increased Binding to Cellular Fc Receptors

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA·antigen immune complexes are selectively transported across Peyer''s patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (FcαRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.     

Autoproteolytic Activation of ThnT Results in Structural Reorganization Necessary for Substrate Binding and Catalysis

cis-Autoproteolysis is a post-translational modification necessary for the function of ThnT, an enzyme involved in the biosynthesis of the β-lactam antibiotic thienamycin. This modification generates an N-terminal threonine nucleophile that is used to hydrolyze the pantetheinyl moiety of its natural substrate. We determined the crystal structure of autoactivated ThnT to 1.8 Å through X-ray crystallography. Comparison to a mutationally inactivated precursor structure revealed several large conformational rearrangements near the active site. To probe the relevance of these transitions, we designed a pantetheine-like chloromethyl ketone inactivator and co-crystallized it with ThnT. Although this class of inhibitor has been in use for several decades, the mode of inactivation had not been determined for an enzyme that uses an N-terminal nucleophile. The co-crystal structure revealed the chloromethyl ketone bound to the N-terminal nucleophile of ThnT through an ether linkage, and analysis suggests inactivation through a direct displacement mechanism. More importantly, this inactivated complex shows that three regions of ThnT that are critical to the formation of the substrate binding pocket undergo rearrangement upon autoproteolysis. Comparison of ThnT with other autoproteolytic enzymes of disparate evolutionary lineage revealed a high degree of similarity within the proenzyme active site, reflecting shared chemical constraints. However, after autoproteolysis, many enzymes, like ThnT, are observed to rearrange in order to accommodate their specific substrate. We propose that this is a general phenomenon, whereby autoprocessing systems with shared chemistry may possess similar structural features that dissipate upon rearrangement into a mature state.     

Ligand-mimicking Receptor Variant Discloses Binding and Activation Mode of Prolactin-releasing Peptide

The prolactin-releasing peptide receptor and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the ligand binding mode of peptide G-protein-coupled receptors (GPCRs). By receptor mutagenesis, we identified the conserved aspartate in the upper transmembrane helix 6 (Asp(6.59)) of the receptor as the first position that directly interacts with arginine 19 of the ligand (Arg(19)). Replacement of Asp(6.59) with Arg(19) of PrRP20 led to D6.59R, which turned out to be a constitutively active receptor mutant (CAM). This suggests that the mutated residue at the top of transmembrane helix 6 mimics Arg(19) by interacting with additional binding partners in the receptor. Next, we generated an initial comparative model of this CAM because no ligand docking was required, and we selected the next set of receptor mutants to find the engaged partners of the binding pocket. In an iterative process, we identified two acidic residues and two hydrophobic residues that form the peptide ligand binding pocket. As all residues are localized on top or in the upper part of the transmembrane domains, we clearly can show that the extracellular surface of the receptor is sufficient for full signal transduction for prolactin-releasing peptide, rather than a deep, membrane-embedded binding pocket. This contributes to the knowledge of the binding of peptide ligands to GPCRs and might facilitate the development of GPCR ligands, but it also provides new targeting of CAMs involved in hereditary diseases.     

Metabolism and Metal Binding by Surface-Colonizing Bacteria: Results of Microgradient Measurements

Short-term (65-h) bacterial colonization of 0.2-μm (pore size) filters submerged in water from Lake Charlotte, Nova Scotia, was characterized by a well-defined succession of cell types, in which small cocci gave way to larger, rod-shaped cells. This succession agrees with the concept of attachment as a strategy for survival, in which inactive cocci can attach to a surface and grow into larger, rod-shaped cells by using endogenous nutrients and the nutrients accumulated at the solid-liquid interface. Analyses of oxygen and CO₂ microgradients above colonized surfaces indicated that a peak of respiration accompanied the succession of rods from cocci. CO₂ fixation then became apparent as the rods began to bind manganese and iron to their surfaces. This means that survival by attachment may not be just the province of heterotrophs. It could also be a strategy adopted by metal-oxidizing chemoautotrophs. Long-term (34-day) colonization of similar filters indicated that, while a succession of attached cell types may indeed be a natural occurrence, other factors (such as the selective grazing of larger cells) tend to obscure the development of this succession.     

Structure of Osh3 Reveals a Conserved Mode of Phosphoinositide Binding in Oxysterol-Binding Proteins

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Influence of Drug Binding on DNA Flexibility: A Normal Mode Analysis

Abstract

DNA-drug complexes are important because of their pharmacological interest but, in addition, they provide a useful model to study the essential aspects of DNA recognition processes. In order to investigate the influence of ligand binding on the dynamic properties of DNA we have carried out normal mode analysis for complexes with drugs of two types: a typical intercalator, 9-aminoacridine, and a typical groove binder, netropsin. Normal modes are analysed in terms of helicoidal parameter variations with special attention being paid to global deformations of the double helix. The results show that the influence of these two drugs is very different. Intercalation of 9-aminoacridine leads to an increase in the flexibility of the intercalated dinucleotide step, with notably larger vibrational amplitudes for both roll and twist parameters compared to free DNA. In contrast, the groove binding of netropsin induces a stiffening of the DNA segment which is in contact with the drug reflected by decreased vibrational amplitudes for backbone angles and inter base pair helicoidal parameters and an increase in vibrations for adjacent base pairs in terms of buckle and propeller twist.     

The Binding Mode of Human Nucleoside Diphosphate Kinase B to Single-Strand DNA

In this paper, we studied the interaction of the human isoform B of nucleoside diphosphatekinase (NDP kinase B) with the nuclease hypersensitive element (NHE) present in the promoterelement of the c-myc oncogene. The DNA-binding properties of NDP kinase B and otherNDP kinases are compared and the nucleotide requirement for binding are discussed. Usingquantitative methods, we identified the DNA-binding sites on the protein and we proposed astructural model for a complex of one hexameric NDP kinase B with an oligonucleotide.     

Kinetics of Ligand-Receptor Interaction Reveals an Induced-Fit Mode of Binding in a Cyclic Nucleotide-Activated Protein

Many receptors and ion channels are activated by ligands. One key question concerns the binding mechanism. Does the ligand induce conformational changes in the protein via the induced-fit mechanism? Or does the protein preexist as an ensemble of conformers and the ligand selects the most complementary one, via the conformational selection mechanism? Here, we study ligand binding of a tetrameric cyclic nucleotide-gated channel from Mesorhizobium loti and of its monomeric binding domain (CNBD) using rapid mixing, mutagenesis, and structure-based computational biology. Association rate constants of ∼10⁷ M⁻¹ s⁻¹ are compatible with diffusion-limited binding. Ligand binding to the full-length CNG channel and the isolated CNBD differ, revealing allosteric control of the CNBD by the effector domain. Finally, mutagenesis of allosteric residues affects only the dissociation rate constant, suggesting that binding follows the induced-fit mechanism. This study illustrates the strength of combining mutational, kinetic, and computational approaches to unravel important mechanistic features of ligand binding.     

Benzodiazepine Receptor Binding Following Amygdala-Kindled Convulsions: Differing Results in Washed and Unwashed Brain Membranes

The binding of [3H]flunitrazepam and [3H]RO5-4864 was measured in unwashed brain homogenates and in extensively washed brain membranes from amygdala-kindled and "yoked" control rats sacrificed 2 weeks following the sixth stage 5 convulsion. In unwashed homogenates, [3H]flunitrazepam binding was reduced in both the hypothalamus and ipsilateral right cortex of kindled rats (unchanged in other areas). In washed brain membranes, [3H]flunitrazepam binding was unaltered in these regions; it was bilaterally elevated, however, in both the amygdala and hippocampus (unchanged in other areas). In washed membranes, the in vitro addition of gamma-aminobutyric acid enhanced [3H]flunitrazepam binding to a similar extent in kindled and control membranes. These data indicate that the type of benzodiazepine binding abnormality observed after kindling depends on the type of tissue preparation employed in the assay procedure.     

Distinct ETA Receptor Binding Mode of Macitentan As Determined by Site Directed Mutagenesis

The competitive endothelin receptor antagonists (ERA) bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min) occupancy half-lives at the ET_A receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ET_A receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ET_A receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ET_A receptor-antagonist interaction modes, we performed functional studies using ET_A receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ET_A receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that – in contrast to bosentan and ambrisentan - macitentan-ET_A receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan''s sustained target occupancy and insurmountable antagonism.     

Novel Clostridium thermocellum Type I Cohesin-Dockerin Complexes Reveal a Single Binding Mode

Protein-protein interactions play a pivotal role in a large number of biological processes exemplified by the assembly of the cellulosome. Integration of cellulosomal components occurs through the binding of type I cohesin modules located in a non-catalytic molecular scaffold to type I dockerin modules located at the C terminus of cellulosomal enzymes. The majority of type I dockerins display internal symmetry reflected by the presence of two essentially identical cohesin-binding surfaces. Here we report the crystal structures of two novel Clostridium thermocellum type I cohesin-dockerin complexes (CohOlpC-Doc124A and CohOlpA-Doc918). The data revealed that the two dockerins, Doc918 and Doc124A, are unusual because they lack the structural symmetry required to support a dual binding mode. Thus, in both cases, cohesin recognition is dominated by residues located at positions 11, 12, and 19 of one of the dockerin binding surfaces. The alternative binding mode is not possible (Doc918) or highly limited (Doc124A) because residues that assume the critical interacting positions, when dockerins are reoriented by 180°, make steric clashes with the cohesin. In common with a third dockerin (Doc258) that also presents a single binding mode, Doc124A directs the appended cellulase, Cel124A, to the surface of C. thermocellum and not to cellulosomes because it binds preferentially to type I cohesins located at the cell envelope. Although there are a few exceptions, such as Doc918 described here, these data suggest that there is considerable selective pressure for the evolution of a dual binding mode in type I dockerins that direct enzymes into cellulosomes.