Molecular modeling study on the enantioselective binding of S- and R-ofloxacin to various DNA sequences

The complex formation of S- and R-ofloxacin with the self-complementary oligonucleotides, namely d[ATAGCGCTAT](2), d[GCGATATCGC](2) and d[ATAICICTAT](2), were investigated by the molecular dynamics (MD) simulation. Four starting positions, including two intercalation positions with different insertion directions and two minor groove binding positions, were considered. The total energy of both S- and R-ofloxacin-d[ATAGCGCTAT](2) complex, in which ofloxacin binds in the minor groove of the oligonucleotide, were lower than any intercalation binding mode. For both enantiomers, formation of the complex with GC oligonucleotide is more favorable than AT and IC oligonucleotides. When S- and R-ofloxacin are compared, the S-enantiomer exhibits more favorable total energy and torsion angles in the complex formation. This result is in agreement with the experimental observation [Hwangbo et al., Eur J Pharm Sci 18, 197 (2003)]. In the complex, both enantiomers form two hydrogen bonds: one between the carbonyl group of ofloxacin and the amine group of G16 and the other between the fluorine group and the G6 amine for S-ofloxacin. However, only one hydrogen bond is formed between endocyclic hydrogen atom at the C2 position of adenine and inosine base and carbonyl group of ofloxacin, which may be the reason for the GC preferentiality of ofloxacin.     

Quantitative analysis of the interaction between S-100 proteins and brain tubulin

S-100 was shown to regulate the in vitro assembly of brain microtubule proteins (MTPs) in a Ca2+-mediated way by acting on both the nucleation and the elongation of microtubules (MTs). Here data will be shown suggesting that S-100 binds to tubulin. The binding is time-, temperature-, Ca2+-, and pH-dependent, and saturable with respect to S-100. At pH 6.75, the saturation curve is biphasic, displaying a high affinity component (dissociation constant, Kd1, approximately 0.1 microM) and a low affinity component (Kd2 approximately 3.8 microM). At pH 6.75, as the free Ca2+ concentration raises from 0 to 100 microM, the overall binding capacity increases from 0.065 to 0.66 mol S-100/mol tubulin dimer. This finding, together with the observation that the S-100 effect on MTP assembly is Ca2+-dependent at that pH, suggests that the S-100-induced inhibition of MTP assembly depends on S-100 binding to the low affinity sites on the tubulin molecule. The S-100 binding to tubulin is pH-dependent; as the pH raises from 6.75 to 8.3, both binding components are affected, the major changes consisting of an increase in the binding capacity and a decrease in the overall affinity. Moreover, as the pH raises, Ca2+ is no longer required for S-100 to bind to tubulin. S-100 also interacts with a component of whole MTPs (probably tubulin, on the basis of the above results). No S-100 binding to microtubule-associated proteins (MAPs) could be evidenced by the techniques employed in this study. On the contrary, some competition between S-100 and MAPs for binding sites or tubulin seems to occur.     

Somatostatin receptors on rat pancreatic acinar cells. Pharmacological and structural characterization and demonstration of down-regulation in streptozotocin diabetes

The binding of somatostatin-14 (S-14) to rat pancreatic acinar cell membranes was characterized using [125I-Tyr11]S-14 as the radioligand. Maximum binding was observed at pH 7.4 and was Ca2+-dependent. Such Ca2+ dependence of S-14 receptor binding was not observed in other tissues. Scatchard analysis of the competitive inhibition by S-14 of [125I-Tyr11]S-14 binding revealed a single class of high affinity sites (Kd = 0.5 +/- 0.07 nM) with a binding capacity (Bmax) of 266 +/- 22 fmol/mg of protein. [D-Trp8]S-14 and structural analogs with halogenated Trp moiety exhibited 2-32-fold greater binding affinity than S-14, [D-F5-Trp8]S-14 being the most potent. [Tyr11]S-14 was equipotent with S-14. The affinity of somatostatin-28 for binding to these receptors was 50% of that of S-14. Cholecystokinin octapeptide (CCK-8) inhibited the binding of [125I-Tyr11]S-14, but its inhibition curve was not parallel to that of S-14. In the presence of 1 nM CCK-8, the Bmax of S-14 receptors was reduced to 150 +/- 17 fmol/mg of protein. Dibutyryl cyclic GMP, a CCK receptor antagonist, partially reversed the inhibitory action of CCK-8, suggesting that CCK receptors mediate the inhibition of S-14 receptor binding. GDP, GTP, and guanyl-5'-yl imidodiphosphate inhibit S-14 receptor binding in this tissue. The inhibition was shown to be due to decrease in binding capacity and not due to change in affinity. Specifically bound [125I-Tyr11]S-14 cross-linked to the S-14 receptors was found associated with three proteins of approximate Mr = 200,000, 80,000, and 70,000 which could be detected under both reducing and nonreducing conditions. Finally, pancreatic acinar cell S-14 receptors were shown to be down-regulated by persistent hypersomatostatinemia 1 week after streptozotocin-induced diabetes characterized by decreased Bmax (105 +/- 13 fmol/mg of protein) without any change in affinity. We conclude that pancreatic acinar cell membrane S-14 receptors require Ca2+ for maximal binding and thus differ from S-14 receptors in other tissues, S-14 receptors in this tissue also exhibit selective ligand specificities, these receptors are regulated by CCK-8 and guanine nucleotides, three receptor proteins of apparent Mr = 200,000, 80,000, and 70,000 specifically bind S-14, and (v) these receptors are regulated by S-14 in vivo as evidenced by decreased binding in streptozotocin diabetic rats characterized by hypersomatostatinemia.     

Calcium binding and calcium-sensitivity of heavy meromyosin and subfragment-1 from squid (Todarodes pacificus) mantle and scallop (Patinopecten yessoensis) adductor muscles

1. HMM and S-1 both bind one mol of calcium per mole of head, and a half of the calcium binding was diminished upon magnesium addition (10 mM) at the low affinity site. 2. The Mg-ATPase activity of HMM (without actin) was fully activated by the binding of one mol of calcium bound per mol of HMM. 3. The calcium binding profile to S-1 is the same as that to HMM, however, the Mg-ATPase activity of S-1 is independent of calcium binding. It is suggested that there are two kinds of myosin head (or S-1) in molluscan myosin, functionally different in calcium binding properties.     

ST-1, a 39-kilodalton protein in Trypanosoma brucei, exhibits a dual affinity for the duplex form of the 29-base-pair subtelomeric repeat and its C-rich strand.

In our attempt to identify telomere region-binding proteins in Trypanosoma brucei, we identified ST-1, a polypeptide with novel features. ST-1 was chromatographically purified from S-100 cell extracts and was renatured from a sodium dodecyl sulfate-protein gel as a 39-kDa polypeptide. It forms a specific complex with the trypanosome telomere repeats of TTAGGG, but more significantly, it shows a higher affinity for the 29-bp subtelomere repeats of T. brucei. These 29-mer boxes are a large tandem series of telomere-derived repeats which separate the simple telomere DNA from middle-repetitive telomere-associated sequences on many chromosomes. ST-1 is the first example of a protein binding within such large repetitive subtelomere elements in trypanosomes or other organisms. ST-1 is also novel in that it has a selective affinity for the C-rich strands of both the subtelomeric 29-mer and the telomere repeats, comparable to that for the duplex form of the respective repeats. All previously described telomere-binding proteins have affinity for only the duplex form or for the G-rich strand. This C-rich strand binding specificity of ST-1 may provide insight into this protein's mechanism of binding in vivo.     

G-quadruplex DNA aptamers generated for systemin

Ligands specific to bioactive molecules play important roles in biomedical researches and applications, such as biological assay, diagnosis and therapy. Systemin is a peptide hormone firstly identified in plant. In this paper we report the selection of a group of DNA aptamers that can specifically bind to systemin. Through comparing the predicted secondary structures of all the aptamers, a hairpin structure with G-rich loop was determined to be the binding motif of these aptamers. The G-rich loop region of this binding motif was further characterized to fold into an antiparallel G-quadruplex by truncation-mutation assay and CD spectrum. The apparent equilibrium dissociation constant (K(d)) of one strong binding sequence (S-5-1) was measured to be 0.5 μM. The specificity assay shows that S-5-1 strongly bind to whole systemin, weakly bind to truncated or mutated systemin and does not bind to the scrambled peptide with the same amino acid composition as systemin. The high affinity and specificity make S-5-1 hold potentials to serve as a molecular ligand applied in detection, separation and functional investigation of systemin in plants.     

Regional serotonin receptor studies: Chronic methysergide treatment induces a selective and dose-dependent decrease in serotonin-2 receptors in mouse cerebral cortex

Compared with the well described supersensitization responses of dopaminergic and beta-adrenergic receptors to pharmacologic antagonists and denervation, the regulation of serotonin-1 (S-1) and serotonin-2 (S-2) receptors is poorly understood. In an effort to modulate S-1 and S-2 receptors in mouse brain, male C57BL/6J mice were treated chronically with methysergide, a serotonin antagonist with nanomolar affinity for both S-1 and S-2 receptors. Methysergide treatment had no influence on the affinity or density of S-1 receptors as measured by binding of (³H)-5-HT in cerebral cortex, hippocampus or hypothalamus. In contrast, the S-2 receptor specific binding of (³H)-spiperone in the cerebral cortex decreased in a dose dependent fashion, a direction of change opposite to that usually seen in catecholamine pathways chronically exposed to antagonists. The effect was selective for the S-2 serotonergic receptor since the D-2 dopaminergic receptor specific binding of (³H)-spiperone in the caudate nucleus was unaffected by drug treatment. These results suggest that either serotonin receptors respond atypically to chronic receptor blockade by antagonist or that in vivo, methysergide may have additional pre-synaptic effects on serotonin uptake or release.     

On the binding of L-S- and L-R-diastereoisomers of the substrate analog L-methionine sulfoximine to glutamine synthetase from Escherichia coli

Active site ligand interactions with dodecameric glutamine synthetase from Escherichia coli were studied spectrally, using the resolved L-S- and L-R-diastereoisomers of the substrate analog L-methionine-SR-sulfoximine. direct measurements of the reversible binding of the S-isomer to unadenylylated manganese-enzyme show a stoichiometry of 1 eq/subunit and negative cooperativity with a Hill coefficient of 0.7. The affinity of this enzyme complex is greatest for the S-isomer alone ([S]0.5 = 35 microM), least with the R-isomer alone ([S]0.5 = 0.38 mM), and intermediate (but closer to that for the S-isomer) for an equimolar mixture of S- and R-isomers ([S]0.5 = 61 microM). The affinity for the S-isomer is enhanced greater than 35-fold by ADP and is decreased approximately 3-fold by adenylylation of the enzyme. Shrake, A., Whitley, E. J. Jr., and Ginsburg, A. ((1980) J. Biol. Chem. 255, 581-589) reported that UV spectral perturbations markedly differ for binding commercial L-methionine-SR-sulfoximine to unadenylylated and adenylylated manganese enzymes. However, essentially the same saturating protein difference spectrum is produced by binding the resolved S- and R-diastereoisomers, and equimolar mixture of S- and R-isomers, and the commercial S- and R-isomeric mixture to a particular enzyme complex. Since neither the subunit interactions that give rise to the observed negative cooperativity of binding nor the affinity differences in binding the S- and R-isomers are reflected in protein difference spectra, spectral perturbations derive from a conformational change that is solely a marked for the occupancy of the single subunit site by either isomer.     

S-100 proteins

S-100 is a group of closely related, small, acidic Ca2+-binding proteins (S-100a0, S-100a and S-100b, which are alpha alpha, alpha beta, and beta beta in composition, respectively). S-100 is structurally related to calmodulin and other Ca2+-binding proteins. S-100 is abundant in the brain and is contained in well defined cell types of both neuroectodermal and non-neuroectodermal origin, as well as in their neoplastic counterparts. In the mammalian brain, S-100a and S-100b are confined to glial cells, while S-100a0 is neuronal in localization. Single S-100 isoforms bind Ca2+ with nearly the same affinity. K+ antagonizes the binding of Ca2+ to high affinity sites on S-100. S-100 binds Zn2+ with high affinity. S-100 is found in a soluble and a membrane-bound form and has the ability to interact with artificial and natural membranes. S-100 has no enzymatic activity. S-100 has been involved in several activities including memory processes, regulation of diffusion of monovalent cations across membranes, modulation of the physical state of membranes, regulation of the phosphorylation of several proteins, control of the assembly-disassembly of microtubules. Some of these effects are strictly Ca2+-dependent, while other are not. S-100 is being secreted or released to the extracellular space. In some cases, this event is hormonally regulated. Several S-100 binding proteins are being described.     

Effect of nucleotide on the binding of N,N'-p-phenylenedimaleimide-modified S-1 to unregulated and regulated actin

In our previous study [Chalovich, J. M., Greene, L. E., & Eisenberg, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4909-4913], myosin subfragment 1 that was modified by having its two reactive thiol groups cross-linked by N,N'-p-phenylenedimaleimide (pPDM) was found to resemble the myosin subfragment 1-adenosine 5'-triphosphate (S-1.ATP) complex in its interaction with actin. In the present study, we examined the effect of actin on adenosine 5'-diphosphate (ADP) trapped at the active site of pPDM.S-1. Our results indicate first that, in the presence of actin, ADP is no longer trapped at the active site but exchanges rapidly with free nucleotide. Different pPDM.S-1.nucleotide complexes were then formed by exchanging nucleotide into the active site of pPDM.S-1 in the presence of actin. The binding of pPDM.S-1.ATP or pPDM.S-1.PPi to actin is virtually identical with that of unmodified S-1 in the presence of ATP. Specifically, at mu = 18 mM, 25 degrees C, pPDM.S-1.ATP or pPDM.S-1.PPi binds to unregulated actin with the same affinity as does S-1.ATP, and this binding does not appear to be affected by troponin-tropomyosin. On the other hand, pPDM.S-1.ADP and pPDM.S-1 with no bound nucleotide both show a small, but significant, difference between their binding to actin and the binding of S-1.ATP; pPDM.S-1 and pPDM.S-1.ADP both bind about 2- to 3-fold more strongly to unregulated actin than does S-1.ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subnuclear Distribution of the S-100 Protein Specific Binding Sites in Rat Brain

Abstract: Fractionation of isolated brain nuclei previously reacted with ¹²⁵I-labelled S-100 showed that most of the specifically bound radioactivity associated with the nuclear membranes and the nucleoli. Labelling of nucleoli, which indicates the entrance of ¹²⁵I-labelled S-100 into the nucleus, was observed at 37°C, but not at 0–4°C. When tested separately for ¹²⁵I-labelled S-100 specific binding, both the nuclear membranes and the nucleoli were found to bind ¹²⁵I-labelled S-100 in a biphasic manner, the binding displaying a high affinity and a low affinity component, as observed with intact nuclei. However, the binding to nuclear membranes was largely irreversible, while that to nucleoli was fully reversible after any association time.     

The hRad51 and RecA proteins show significant differences in cooperative binding to single-stranded DNA.

The human Rad51 protein (hRad51), like its bacterial homologue RecA, catalyzes genetic recombination between homologous single and double-stranded DNA substrates. Using IAsys biosensor technology, we have examined the critical first step in this process, the binding of hRad51 and RecA to ssDNA. We show that hRad51 binds cooperatively and with high affinity to an oligonucleotide substrate in both the absence and presence of nucleotide cofactors. In fact, both ATP and ATPgammaS have a slight inhibitory effect on hRad51 binding affinity. We show that this results from a decrease in the intrinsic affinity of a given monomer for ssDNA, which is counterbalanced by an increase in the cooperative assembly of protein onto DNA. In contrast, we show that the dramatic NTP-induced increase in ssDNA binding affinity of RecA is accounted for by a significant increase in cooperative filament assembly and not by an increase in the intrinsic DNA binding affinity of monomeric RecA. These results demonstrate that although the hRad51 and RecA proteins display many structural and functional similarities, they show profound inherent mechanistic differences.     

Analysis of p53 "latency" and "activation" by fluorescence correlation spectroscopy. Evidence for different modes of high affinity DNA binding

The concept that the tumor suppressor p53 is a latent DNA-binding protein that must become activated for sequence-specific DNA binding recently has been challenged, although the "activation" phenomenon has been well established in in vitro DNA binding assays. Using electrophoretic mobility shift assays and fluorescence correlation spectroscopy, we analyzed the binding of "latent" and "activated" p53 to double-stranded DNA oligonucleotides containing or not containing a p53 consensus binding site (DNAspec or DNAunspec, respectively). In the absence of competitor DNA, latent p53 bound DNAspec and DNAunspec with high affinity in a sequence-independent manner. Activation of p53 by the addition of the C-terminal antibody PAb421 significantly decreased the binding affinity for DNAunspec and concomitantly increased the binding affinity for DNAspec. The net result of this dual effect is a significant difference in the affinity of activated p53 for DNAspec and DNAunspec, which explains the activation of p53. High affinity nonspecific DNA binding of latent p53 required both the p53 core domain and the p53 C terminus, whereas high affinity sequence-specific DNA binding of activated p53 was mediated by the p53 core domain alone. The data suggest that high affinity nonspecific DNA binding of latent and high affinity sequence-specific binding of activated p53 to double-stranded DNA differ in their requirement for the C terminus and involve different structural features of the core domain. Because high affinity nonspecific DNA binding of latent p53 is restricted to wild type p53, we propose that it relates to its tumor suppressor functions.     

The influence of DNA binding protein on the substrate affinities of DNA polymerase from Ustilago maydis: One polymerase implicated in both DNA replication and repair

Summary The DNA polymerase of Ustilago maydis is stimulated by a DNA binding protein from the same organism. Analysis of this stimulation shows that there is an increase in affinity for both substrates of the reaction. The apparent Km for deoxynucleoside triphosphates is decreased 3 fold, and that for denatured DNA by 4 fold. In both cases the maximum velocity (Vmax) is increased 1.2 to 1.4 fold. It is suggested that the variability in the affinity of the enzyme for deoxynucleoside triphosphates mediated by the binding protein may provide the basis for the UV sensitivity of pyrimidine auxotrophs in this organism.     

Binding of ADP and ATP analogs to cross-linked and non-cross-linked acto X S-1

We previously determined the binding constants of ADP, adenylyl imidodiphosphate (AMP-PNP), and inorganic pyrophosphate (PPi) to acto . myosin subfragment 1 (acto X S-1) by measuring the dissociation of acto X S-1 as a function of ATP analog concentration (Greene, L.E., and Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548). In the present study, we reinvestigated this question by measuring the extent to which these ATP analogs inhibit the acto X S-1 ATPase activity using both cross-linked actin X S-1 and non-cross-linked proteins. No significant difference was found between the cross-linked and non-cross-linked acto X S-1 complexes in their affinity for either ADP or AMP-PNP. The binding constant of ADP to acto X S-1 determined by the inhibition method was in excellent agreement with that obtained previously by the dissociation method, both techniques giving values of about 7 X 10(3) M-1. However, this was not the case for AMP-PNP and PPi, with the inhibition method giving about 10-fold weaker binding constants than those determined previously by the dissociation method. Upon redoing our dissociation experiments over a wider range of actin concentrations than we used previously, we now find that the dissociation method gives much weaker values for the binding constants of PPi and AMP-PNP to acto X S-1, i.e. values on the order of 4 X 10(2) M-1. The very weak binding of these ATP analogs to acto X S-1 makes it difficult to obtain these values with great accuracy. Nevertheless, they seem to be in good agreement with the binding constants determined by the inhibition method. The weak binding of AMP-PNP and PPi to acto X S-1 is consistent with the recent fiber studies of Pate and Cooke (Pate, E., and Cooke, R. (1985) Biophys. J. 47, 773-780) and Schoenberg and Eisenberg (Schoenberg, M., and Eisenberg, E. (1986) Biophys. J. 48, 863-872).     

Purification of the thromboxane A2/prostaglandin H2 receptor from human blood platelets

S-145 (5Z-7-(3-endo-phenylsulfonylamino-(2.2.1.)-bicyclohept -2-exo-yl) heptenoic acid) is a potent and selective antagonist for thromboxane A2/prostaglandin H2 receptor. Using this compound as an immobilized ligand for affinity chromatography and [3H]S-145 as a radioligand, we have purified the thromboxane A2/prostaglandin H2 receptor from the membranes of human blood platelets. The purification procedures consisted of solubilization of the receptor with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), affinity chromatographies on columns of S-145 affinity gel, wheat germ agglutinin agarose and red agarose, and repeated gel filtration high performance liquid chromatography on a TSK gel G-3000SW column. On the second gel filtration high performance liquid chromatography, the [3H]S-145 binding activity was eluted as a symmetrical peak which overlapped exactly with a peak of ultraviolet absorption at 280 nm. By these procedures, the receptor was purified about 8700-fold from the solubilized extract with a recovery of 6%. The final preparation showed a broad protein band at Mr 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maximally bound 19.2 nmol of [3H]S-145/mg protein with a Kd of 29.8 nM. The [3H]S-145 binding to the purified receptor was specifically displaced by several thromboxane A2/prostaglandin H2 analogues.     

DNA bending and twisting properties of integration host factor determined by DNA cyclization

The binding of many proteins to DNA is profoundly affected by DNA bending, twisting, and supercoiling. When protein binding alters DNA conformation, interaction between inherent and induced DNA conformation can affect protein binding affinity and specificity. Integration host factor (IHF), a sequence-specific, DNA-binding protein of Escherichia coli, strongly bends the DNA upon binding. To assess the influence of inherent DNA bending on IHF binding, we took advantage of the high degree of natural static curvature associated with an IHF site on a 163-bp minicircle and measured the binding affinity of IHF for its recognition site contained on this DNA in both circular and linear form. IHF showed a higher affinity for the circular form of the DNA when compared to the linear form. In addition, the presence of IHF during DNA cyclization changed the topology of cyclization products and their ability to bind IHF, consistent with IHF untwisting DNA. These results show that inherent DNA conformation anisotropy is an important determinant of IHF binding affinity and suggests a mechanism for modulation of IHF activity by local DNA conformation.     

Interaction Between S-100 Proteins and Steady-State and Taxol-Stabilized Microtubules In Vitro

S-100 proteins are a group of three 21-kilodalton, acidic, Ca2+-binding proteins of the "E-F hand" type shown to regulate several cell activities, including microtubule (MT) assembly-disassembly. We show here that S-100 proteins interact with MTs assembled from either whole microtubule protein or purified tubulin, both in the absence and in the presence of the MT-stabilizing drug taxol. Evidence for the binding of S-100 to MTs comes from both kinetic (turbidimetric) and binding studies. Kinetically, S-100 enhances the disassembly of steady-state MTs in the presence of high concentrations of colchicine or vinblastine at 10 microM free Ca2+ and disassembles taxol-stabilized MTs at high Ca2+ concentrations. Experiments performed using 125I-labeled S-100 show that S-100 binds Ca2+ independently to a single set of sites on taxol-stabilized MTs assembled from pure tubulin with an affinity of 6 x 10(-5) M and a stoichiometry of 0.15 mol of S-100/mol of polymerized tubulin. Under certain conditions, S-100 proteins also cosediment with MTs prepared by coassembly of S-100 with MTs, probably in the form of an S-100-tubulin complex. Because S-100 binds to MTs under conditions where this protein fraction does not produce observable effects on the kinetics of assembly-disassembly, e.g., in the absence of Ca2+ at pH 6.7, we conclude that the S-100 binding to MTs does not affect the stability of MTs per se, but rather creates conditions for increased sensitivity of MTs to Ca2+.     

Maize Mitochondrial Plasmid S-1 Sequences Share Homology with Chloroplast Gene psbA

The linear, 6397-base pair (bp), mitochondrial S-1 DNA molecule from maize contains a 420-bp segment that is homologous with the chloroplast gene (psbA) that codes for the quinone binding protein of photosystem II. This is the first report of a chloroplast sequence in a naturally occurring viral-like or plasmid DNA. The complete sequence of the S-1 chloroplast segment has been compared with homologous regions of six different chloroplast genes. The S-1 segment has diverged from the other genes both by length mutation and base substitution. Several of the length mutations are exact adjacent tandem duplications of 4 and 5 bp similar to "footprints" left after excision of transposable elements in maize nuclear DNA.