Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Double Bind Interaction”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources
PubMed1993

Influence of DNA base sequence on the binding energetics of actinomycin D.

The influences of base sequence on the thermodynamic properties associated with the interaction of actinomycin D with DNA are examined. It has been previously established that GpC steps of double-helical DNAs are highly preferred binding sites for actinomycin D. In this study, a series of oligonucleotides was designed and synthesized to probe the effects of flanking base sequence (adjacent to the GpC step) and novel non-GpC binding sites on the binding of actinomycin D. The use of these oligonucleotides provides a direct method for quantitating sequence specificities and actinomycin D binding energetics. Effects of different 5' and 3' flanking nucleotides on the interactions of actinomycin with the core GpC binding sites were examined using UV-visible spectrophotometric methods, and changes in binding energetics were quantitated. These studies demonstrate strong actinomycin D binding affinities to both classical GpC and an atypical non-GpC site. Enthalpy and entropy components of the DNA binding energetics for the GpC binding sites are compared and correlated with those determined for actinomycin D binding to the high-affinity non-GpC site of an 11-mer containing TGGGT as the central sequence. This TGGGT site, first suggested to be a high-affinity sequence in our earlier photoaffinity labeling studies, exhibits binding of actinomycin D comparable in strength to that of traditional actinomycin D binding sites (i.e., GpC steps). From these studies, the overall affinity and specific thermodynamic contributions (delta H degree, delta S degree) to binding of actinomycin D are demonstrated to be highly influenced both by the sequence at the intercalation site and by neighboring bases which flank the intercalation site.

Amino Acid Sequence↗
PubMed1997

Residues at the carboxy terminus of T4 DNA polymerase are important determinants for interaction with the polymerase accessory proteins.

Three T4 DNA polymerase accessory proteins (44P/62P and 45P) stimulate the polymerase (pol) activity and the 3'-5' exonuclease (exo) activity of T4 DNA polymerase (43P) on long, double-stranded DNA substrates. The 44P/62P "clamp loader" facilitates the binding of 45P, the "sliding clamp", to DNA that is primed for replication. Using a series of truncated 43P mutants, we identified a region at the extreme carboxy terminus of the DNA polymerase that is required for its interaction with accessory proteins. Truncation mutants of 43P lacking the carboxy-terminal 3, 6, or 11 residues retained full pol and exo activity on short synthetic primer-templates. However, the ability of the accessory proteins to enhance these activities on long double-stranded DNA templates was drastically reduced, and the extent of the reduction in activity was greater as more residues were deleted. One of the truncation mutants (N881), which had 17 residues removed from the carboxy terminus, showed reduced binding affinity and diminished pol activity but enhanced exo activity upon incubation with a small primer-template. The exo activity of the N881 mutant, on short, single-stranded DNA was unchanged, however, compared to the wild-type enzyme. These results are consistent with inferences drawn from the crystal structure of a DNA polymerase from a related T-even phage, RB69, where the carboxy-terminal 12 residues (equivalent to the 11 residues of 43P from phage T4) protrude from the thumb domain and are free to interact with complementary surfaces of the accessory proteins. The structural integrity of the thumb region in the N881 mutant is probably perturbed and could account for its reduced binding affinity and pol activity when incubated with short, double-stranded DNA substrates.

Amino Acid Sequence↗
PubMed2003

Mutants of protein kinase A that mimic the ATP-binding site of protein kinase B (AKT).

The mutation of well behaved enzymes in order to simulate less manageable cognates is the obvious approach to study specific features of the recalcitrant target. Accordingly, the prototypical protein kinase PKA serves as a model for many kinases, including the closely related PKB, an AGC family protein kinase now implicated as oncogenic in several cancers. Two residues that differ between the alpha isoforms of PKA and PKB at the adenine-binding site generate differing shapes of the binding surface and are likely to play a role in ligand selectivity. As the corresponding mutations in PKA, V123A would enlarge the adenine pocket, while L173M would alter both the shape and its electronic character of the adenine-binding surface. We have determined the structures of the corresponding double mutant (PKAB2: PKAalpha V123A, L173M) in apo and MgATP-bound states, and observed structural alterations of a residue not previously involved in ATP-binding interactions: the side-chain of Q181, which in native PKA points away from the ATP-binding site, adopts in apo double mutant protein a new rotamer conformation, which places the polar groups at the hinge region in the ATP pocket. MgATP binding forces Q181 back to the position seen in native PKA. The crystal structure shows that ATP binding geometry is identical with that in native PKA but in this case was determined under conditions with only a single Mg ion ligand. Surface plasmon resonance spectroscopy studies show that significant energy is required for this ligand-induced transition. An additional PKA/PKB mutation, Q181K, corrects the defect, as shown both by the crystal structure of triple mutant PKAB3 (PKAalpha V123A, L173M, Q181K) and by surface plasmon resonance spectroscopy binding studies with ATP and three isoquinoline inhibitors. Thus, the triple mutant serves well as an easily crystallizable model for PKB inhibitor interactions. Further, the phenomenon of Q181 shows how crystallographic analysis should accompany mutant studies to monitor possible spurious structural effects.

Adenosine Triphosphate↗
PubMed1981

Fluorospectrometric studies on neothramycin and its reaction with DNA.

The characteristics of neothramycin and its reaction mechanism with DNA were studied by fluorescence spectroscopy. The fluorescence intensity of neothramycin is enhanced by the reaction with DNA. The reaction rate of the drug and DNA is rather slow; and is dependent upon both DNA and drug concentrations, and is stimulated by hydrogen ion. Analysis of the equilibrium reaction suggested that neothramycin possesses about one binding site per 2-3 base-pairs with homogeneous affinity and association constant of 4.7 x 10(3) M-1; and no significant mutual interference of the binding sites seems to exist. From the results of neothramycin's interaction with various polynucleotides, it was suggested that a guanine base and a double helix conformation of DNA are required for binding the antibiotic. The current results and previous ones, concerning neothramycin-2'-deoxyguanosine adduct formation, suggested that the antibiotic reacts with 2-amino group of guanine base of DNA by dehydration in the manner of a bimolecular (Sn2ca) equilibrium reaction.

Antibiotics, Antineoplastic↗
PubMed1978

Characterization of ellipticine binding to native calf thymus DNA.

The binding of [14C]ellipticine to native calf thymus DNA was studied using equilibrium dialysis. A Scatchard polt revealed the presence of high-and low-affinity binding sites in DNA, the former having a K of 4.0 X 10(7) M(-1) and an n (saturation limiting of binding) of 0.078 (1mol ellipticine/13 mol of DNA nucleotides). The forces involved in stabilizing the high-affinity binding, which has been equated with intercalative binding, were due to a combination of hydrophobic interactions and hydrogen bonding. Difference spectra of ellipticine in the presence of the polydeoxynucleotides, poly d(A-T) or poly d(G-C), showed that there was no base specificity involved in the high-affinity binding. Ellipticine binding to the low-affinity sites, which has been equated with surface binding, was due primarily to the participation of electrostatic interactions of ellipticine with the anionic phosphate groups on the double helical surface of DNA.

Animals↗
PubMed2000

Mutations within the E2 and NS5A protein in patients infected with hepatitis C virus type 3a and correlation with treatment response.

Defined regions of hepatitis C virus (HCV) envelope 2 (E2), PePHD, and nonstructural 5A (NS5A) protein (PKR-binding domain) have been shown to interact with interferon alfa (IFN-alpha)-inducible double-stranded RNA-activated protein kinase (PKR) in vitro, suggesting a possible mechanism of HCV to evade antiviral effects of IFN-alpha. The clinical correlation between amino acid mutations within the E2 PePHD or the NS5A PKR-binding domain and response to antiviral treatment in HCV-3a-infected patients is unknown. Thirty-three patients infected with HCV-3a isolates were treated with IFN-alpha with or without ribavirin. The carboxyterminal half of E2 and of the NS5A gene were sequenced. Sixteen patients achieved a sustained virological response (SR), 6 patients an end-of-treatment response with relapse thereafter (ETR), and 11 patients were nonresponders (NR). Within the PePHD of the E2 protein 0.5 (range, 0-2) mutations were observed in SR patients, whereas the number of mutations in ETR or NR patients was 0.2 (0-1). Quasispecies analyses showed almost no heterogeneity. The mean number of mutations within the PKR-binding domain of the NS5A protein was 1.6 (range, 0-4) in SR patients, 1 (0-2) in ETR patients, and 1.6 (0-3) in NR patients. Patients with higher numbers of mutations within the E2 or NS5A region showed a trend towards lower pretreatment viremia. Phylogenetic and conformational analyses of E2 or NS5A sequences allowed no differentiation between sensitive and resistant isolates. However, mutations within the E2 PePHD in SR patients were frequent, and hydrophobic mutations within the hydrophilic area of PePHD at codon 668 and 669 were exclusively observed in sustained virological responders.

Adult↗
PubMed2003

Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76.

SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76. The crystal structure of a complex between the C-terminal SH3 domain (SH3C) of Mona/Gads and a SLP-76 peptide has now been solved to 1.7 A. The peptide lacks the canonical SH3 domain binding motif P-x-x-P and does not form a frequently observed poly-proline type II helix. Instead, it adopts a clamp-like shape around the circumfence of the SH3C beta-barrel. The central R-x-x-K motif of the peptide forms a 3(10) helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high affinity. Interestingly, the SH3C displays ion-dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions.

Adaptor Proteins, Signal Transducing↗
PubMed1998

NMR study on the binding of d(GGAAATTTCC)2 with a positively charged pentacosapeptide.

To obtain a better understanding of the electrostatic nature of protein-nucleic acid interactions, we have investigated the interaction of a double-stranded decamer d(GGAAATTTCC)2 with a synthetic arginine and lysine-rich pentacosapeptide (Pep25), using NMR and optical spectroscopy. The chemical shift data of the decamer under various experimental conditions show that the binding of Pep25 changes the conformation of the decamer in a different way, as compared to the conformational changes induced by a variation in temperature or ionic strength. The chemical shift results are interpreted in terms of ring current effects that emerge into a model for the conformational change, in which the double-stranded helix of the decamer undergoes a decrease of twist and rise to accommodate Pep25. The binding results indicate that the positively charged arginine and lysine side chains of Pep25 not only have a stabilising electrostatic interaction with the negatively charged backbone phosphates of d(GGAAATTTCC)2, but also that a stabilisation of the base pairs of d(GGAAATTTCC)2 by Pep25 takes place.

Amino Acid Sequence↗
PubMed1997

Solid-state REDOR NMR distance measurements at the ligand site of a bacterial chemotaxis membrane receptor.

The Escherichia coli serine receptor senses serine levels in the environment and transmits this information across the bacterial inner membrane to modulate a protein phosphorylation cascade which controls swimming behavior. Solid-state nuclear magnetic resonance (NMR) has been used to characterize specific structural features of the ligand binding site interactions in the intact, membrane-bound Ser receptor. Rotational-echo double-resonance (REDOR) experiments on [15N]Ser bound to a [1-13C]Phe-receptor preparation are used to measure distances between the ligand amino group and the carbonyls of two phenylalanine residues in the ligand binding pocket. The results indicate two 4.0 +/- 0.2 A distances, in excellent agreement with the X-ray crystal structure of a soluble fragment of the homologous aspartate receptor [Milburn et al. (1991) Science 254, 1342-1347]. These results confirm the similarity of the binding sites of the Asp and Ser receptors, and demonstrate the feasibility of using solid-state NMR measurements to obtain specific structural information on the 120 kDa intact receptor for probing transmembrane signaling mechanisms.

Bacterial Proteins↗
PubMed1979

Single strand DNA binding of simian virus 40 tumor antigen.

Simian virus 40 T antigen binds to both single and double strand DNA. The single and double strand DNA binding activity of crude T antigen preparations was evaluated by chromatography of the antigen on DNA-cellulose columns. Crude T antigen was retained on both native and denatured DNA-cellulos columns and was eluted from both columns under similar conditions. The interaction of highly purified T antigen with single and double strand DNA was evaluated by competition experiments using a DNA filter binding assay. These experiments showed that T antigen binds preferentially to single strand calf thymus DNA by more than an order of magnitude when compared to double strand calf thymus DNA.

Antigens, Neoplasm↗
PubMed2002

Functional epitope of common gamma chain for interleukin-4 binding.

Interleukin 4 (IL-4) can act on target cells through an IL-4 receptor complex consisting of the IL-4 receptor alpha chain and the common gamma chain (gamma(c)). An IL-4 epitope for gamma(c) binding has previously been identified. In this study, the gamma(c) residues involved in IL-4 binding were defined by alanine-scanning mutational analysis. The epitope comprises gamma(c) residues I100, L102, and Y103 on loop EF1 together with L208 on loop FG2 as the major binding determinants. These predominantly hydrophobic determinants interact with the hydrophobic IL-4 epitope composed of residues I11, N15, and Y124. Double-mutant cycle analysis revealed co-operative interaction between gamma(c) and IL-4 side chains. Several gamma(c) residues involved in IL-4 binding have been previously shown to be mutated in X-linked severe combined immunodeficiency. The importance of these binding residues for gamma(c) function is discussed. These results provide a basis for elucidating the molecular recognition mechanism in the IL-4 receptor system and a paradigm for other gamma(c)-dependent cytokine receptor systems.

Amino Acid Sequence↗
PubMed1999

DNA multimode interaction with berenil and pentamidine; double helix stiffening, unbending and bending.

The antitrypanosomal drugs berenil (Ber) and pentamidine (Pm) preferentially bind to DNA in the minor groove of A.T-rich domains. The properties of A.T clusters are essential for sequence-mediated helix bending. Groove binding drugs locally stiffen the DNA helix but may also change intrinsic helix bends or may bend straight DNA. Ligand binding to randomly distributed sites alters the apparent DNA persistence length, a. Criteria permit the distinction of the underlying mechanism(s). Helix bends, if phased with the helix screw, however, generate solenoidal superhelix components mediating an apparent change of the hydrodynamically effective DNA contour length, L. The measurement of relative changes of both, a and L, as induced by Ber or Pm is performed by titration rotational viscometry. The determination of the two quantities requires two independent measurements: the relative change of DNA intrinsic viscosity, deltay, for short (tending to rod-like) DNA molecules and for comparably long (almost coil-like) ones as a function of r, the bound drug molecules per DNA-P, and this under conditions effectively excluding intramolecular DNA-DNA crosslinking effects. At least at r< or =0.05 and < or =0.03, respectively, the two drugs virtually bind completely to a eukaryotic DNA. r ranges of different drug binding strength and, concomitantly, of different specific conformational response, could be resolved. They represent (sub)modes of different DNA sequences... Whereas the mode-specific elongation effects are fairly similar for both systems, there are pronounced quantitative differences in the relative change of DNA persistence length. The sites of highest Ber-binding strength are correlated to unbent alternating helical A.T segments followed by bent and by less bent or unbent dAn.dTn tracts straightened on Ber-binding. For Pm-DNA interaction the ligand bends the sites of highest Pm affinity. Generally, ligand induced and sequence mediated local DNA-bend removal or DNA bending, as observed for several modes of interaction with A.T rich DNA, are considered to be of gene regulatory relevance.

Animals↗
PubMed2006

Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist binding site.

The insecticide imidacloprid and structurally related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (nAChRs). To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of mutations to basic amino acid residues in loop D of the nAChR acetylcholine (ACh) binding site on the interactions with imidacloprid. The receptors investigated are the recombinant chicken alpha4beta2 nAChR and Drosophila melanogaster Dalpha2/chicken beta2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of Thr77 in loop D of the beta2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the alpha4beta2 nAChR, T77R;E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Likewise, T77K;E79R and T77N;E79R double mutations in the Dalpha2beta2 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of Thr77 to basic residues. By contrast, these double mutations scarcely influenced the ACh concentration-response curve, suggesting selective interactions with imidacloprid of the newly introduced basic residues. Computational, homology models of the agonist binding domain of the wild-type and mutant alpha4beta2 and Dalpha2beta2 nAChRs with imidacloprid bound were generated based on the crystal structures of acetylcholine binding proteins of Lymnaea stagnalis and Aplysia californica. The models indicate that the nitro group of imidacloprid interacts directly with the introduced basic residues at position 77, whereas those at position 79 either prevent or permit such interactions depending on their electrostatic properties, thereby explaining the observed functional changes resulting from site-directed mutagenesis.

Acetylcholine↗
PubMed2003

Genotoxicity studies on DNA-interactive telomerase inhibitors with application as anti-cancer agents.

Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells. However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity. For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102). DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes. The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells. The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis. Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C --> A transversions at position 2 of the missense proline codon of the hisG46 allele. However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells.

Antineoplastic Agents↗
PubMed1999

The recognition of distorted DNA structures by HMG-D: a footprinting and molecular modelling study.

The high mobility group (HMG) domain is a DNA binding motif found in some eukaryotic chromosomal proteins and transcription factors. This domain binds in the minor groove of DNA inducing a sharp bend and also preferentially binds to certain distorted DNA structures. Although structures of sequence-specific HMG domains with their cognate double-helical DNA binding sites have been solved, the nature of the interaction of the domain with distorted DNA remains to be established. In this study we have investigated the interaction of HMG-D, a Drosophila counterpart of the vertebrate HMG1, with a DNA oligomer containing a bulge of two adenine residues. We show by footprinting that HMG-D binds preferentially on one side of the bulged DNA. Based on these data and on the published NMR structures of the HMG domain of HMG-D and the LEF-1-DNA complex, we modelled the HMG-D - bulged DNA complex. This model predicts that two residues, Val32 and Thr33, in the loop between alpha-helices I and II are inserted deep into the "hole" in the DNA formed by the two missing bases on one strand of the DNA bulge. Mutation of these residues confirmed that both are required for the efficient binding and bending of DNA by HMG-D. We discuss both the role of this loop in the recognition of distorted DNA structures by non-sequence specific HMG domain proteins and that of the basic tail in stabilising the induced DNA bend.

Amino Acid Sequence↗
PubMed1997

Electrostatic interactions across a beta-sheet.

The free energy consequences of electrostatic interactions across a model beta-sheet have been probed using a consensus zinc finger peptide. Relative folding free energies have been deduced from coupled peptide folding/metal binding reactions. The energies of the electrostatic interactions have been isolated via double mutant cycles performed at a series of NaCl concentrations. The observed favorable free energies associated with potential ion pairs are modest, less than 0.5 kcal/mol. Unfavorable interactions due to like-charge pairs of similar magnitude were also observed. The largest effects, both favorable and unfavorable, involved interactions with aspartic acid. These observations are consistent with electrostatic interactions occurring without contact ion pair formation with the larger interaction energies involving aspartic acid being due to the relative lack of flexibility of its side chain.

Models, Chemical↗
PubMed1986

Interaction of the tight-binding I12-X86 lac repressor with non operator DNA: salt dependence of complex formation.

The interaction of the wild-type lac repressor and its tight binding double mutant I12-X86 with a non operator-210 base pair-DNA fragment has been investigated using the nitrocellulose filter binding assay. While the affinity of the double mutant for this non specific DNA is increased as compared to that of the wild-type repressor, the number of ions released from the vicinity of the DNA upon complex formation is less important for the mutant than for the wild-type. These results demonstrate that the adaptation in the recognition surface of the repressor recently proposed by Mossing et al (J. Mol. Biol., 1985, 186, 295-305) in the case of an Oc mutant may be a more general phenomenon.

DNA, Bacterial↗
PubMed2002

ET-743: more than an innovative mechanism of action.

Ecteinascidin-743 (ET-743), an anti-tumor agent derived from the marine tunicate, Ecteinascidia turbinata, is active against various solid tumor cell lines, including soft tissue sarcoma, breast, ovarian, non-small-cell lung and prostate cancers and melanoma, and has a broad spectrum of anti-cancer activity in vivo. For reasons as yet unclear, sarcoma cell lines are exquisitely sensitive to ET-743. The drug has a unique mechanism of action that makes it a novel anti-tumor agent. ET-743 is a DNA-binding agent that covalently interacts with the minor groove of the DNA double helix to bend the molecule towards the major groove. Defects in DNA repair pathways have paradoxical effects on the anti-tumor activity of ET-743: loss of mismatch repair does not affect its toxicity; loss of DNA-dependent protein kinase activity enhances its toxicity; defects in transcription-coupled nucleotide excision repair confer resistance to ET-743. As a DNA repair capability appears to be necessary for at least one mechanism of ET-743-mediated cytotoxicity, the drug may interact with the DNA repair machinery to induce lethal strand breaks. One of the most novel aspects of ET-743 is its effect on RNA polymerase II-mediated gene transcription. ET-743 selectively inhibits activation of the multidrug resistance gene, while leaving constitutive gene expression relatively unaffected. Preliminary studies of other genes and transcriptional inducers indicate that ET-743 may be a more general inhibitor of activated, but not basal, transcription.

Antineoplastic Agents, Alkylating↗